Immunogenic wt-1 peptides and methods of use thereof

ABSTRACT

This invention provides peptides, immunogenic compositions and vaccines, and methods of treating, reducing the incidence of, and inducing immune responses to a WT-1-expressing cancer, comprising peptides derived from the WT-1 protein.

GOVERNMENT SUPPORT

This work was supported by grants CA23766, CA59350 and CA08748 from theNational Institutes of Health. The US government has certain rights inthe invention.

FIELD OF INVENTION

This invention provides peptides, compositions and vaccines comprisingsame, and methods of treating, reducing the incidence of, and inducingimmune responses to a WT-1-expressing cancer, comprising administeringsame.

BACKGROUND OF THE INVENTION

Wilms tumor (WT), a pediatric nephroblastoma that occurs with afrequency of 1 in 10,000 births, has been the subject of intenseclinical and basic research for several years. The tumor is embryonic inorigin; it is detected in children usually during the first 5 years oflife and can occur unilaterally or bilaterally. A WT arises whencondensed metanephric mesenchymal cells of the developing kidney fail toproperly differentiate. The implication of the Wilms tumor 1 (WT-1)tumor suppressor gene in the etiology of WT illustrated the impact thatgenetic alterations can have on both development and tumorigenesis.

Wilms tumor protein I (WT-1) is a zinc finger transcription factorexpressed during normal ontogenesis such as in fetal kidney, testis andovary. In adults, WT-1 expression is limited to low levels onhematopoietic stem cells, myoepithelial progenitor cells, renalpodocytes and some cells in testis and ovary. Recent demonstration thatWT-1 is over expressed in several types of leukemia suggested that WT-1would be an attractive target for immunotherapy for various cancers.

SUMMARY OF THE INVENTION

This invention provides peptides, compositions, and immunogeniccompositions such as vaccines comprising immunogenic peptides, andmethods of treating, reducing the incidence of, and inducing immuneresponses to a WT-1-expressing cancer, comprising administeringimmunogenic peptides.

In one embodiment, the present invention provides an isolated WT-1peptide having an amino acid (AA) sequence consisting of any one of thesequences SEQ ID NO:1-160, 162-185, 190, 191 and 193. In one embodiment,the present invention provides an isolated HLA class I binding WT-1peptide having an amino acid (AA) sequence consisting of any one of thesequences SEQ ID NO:142, 143, 144, 145, 146, 147, 148, 149, 150, 151,152, 153, 154, 155, 156, 157, 158, 159, 160, 162, 163, 164, 165, 166,167, 168, 169, 170, 171, 173, 174, 175, 176, 177, 178, 179, 180, 181,182, 183, 184, 185, 190, 191 and 193. In one embodiment, the presentinvention provides an isolated HLA class II binding WT-1 peptide havingan amino acid (AA) sequence consisting of any one of the sequences SEQID NO:149, 156, 173, 174 and 180.

In one embodiment, the present invention provides an isolated WT-1peptide having an amino acid (AA) sequence consisting of any one of thesequences SEQ ID NO: 1-160, 162-185, 190, 191 and 193, or a fragment ofany of the foregoing. In one embodiment, the present invention providesan isolated HLA class I binding WT-1 peptide having an amino acid (AA)sequence consisting of any one of the sequences SEQ ID NO:142, 143, 144,145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158,159, 160, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 173, 174,175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 190, 191 and 193.In one embodiment, the present invention provides an isolated HLA classII binding WT-1 peptide having an amino acid (AA) sequence consisting ofany one of the sequences SEQ ID NO:149, 156, 173, 174 and 180.

In another embodiment, the present invention provides a compositioncomprising (a) an antigen-presenting cell and (b) a peptide selectedfrom SEQ ID NO:1-160, 162-185, 190, 191 and 193. In another embodiment,the present invention provides a composition comprising (a) anantigen-presenting cell and (b) an HLA class I binding peptide selectedfrom SEQ ID NO:142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152,153, 154, 155, 156, 157, 158, 159, 160, 162, 163, 164, 165, 166, 167,168, 169, 170, 171, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182,and 183. In another embodiment, the present invention provides acomposition comprising (a) an antigen-presenting cell and (b) an HLAclass II binding peptide selected from SEQ ID NO:149, 156, 173, 174 and180.

In another embodiment, the present invention provides a vaccinecomprising one or more peptides of SEQ ID NO:1-160, 162-185, 190, 191and 193. In another embodiment, the present invention provides a vaccinecomprising one or more HLA class I binding peptides selected from SEQ IDNO:142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155,156, 157, 158, 159, 160, 162, 163, 164, 165, 166, 167, 168, 169, 170,171, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, and 183. Inanother embodiment, the present invention provides a vaccine comprisingone or more HLA class II binding peptides selected from SEQ ID NO:149,156, 173, 174 and 180. In another embodiment, the present inventionprovides a vaccine comprising one or more HLA class I binding peptidesselected from SEQ ID NO:142, 143, 144, 145, 146, 147, 148, 149, 150,151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 162, 163, 164, 165,166, 167, 168, 169, 170, 171, 173, 174, 175, 176, 177, 178, 179, 180,181, 182, and 183, and one or more HLA class II binding peptidesselected from SEQ ID NO:149, 156, 173, 174 and 180.

In another embodiment, the present invention provides a method oftreating a subject with a WT-1-expressing cancer, the method comprisingadministering to the subject a WT-1peptide or vaccine of the presentinvention, thereby treating a subject with a WT-1-expressing cancer.

In another embodiment, the present invention provides a method ofreducing the incidence of a WT-1-expressing cancer, or its relapse, in asubject, the method comprising administering to the subject a WT-1peptide or vaccine of the present invention, thereby reducing theincidence of a WT-1-expressing cancer, orits relapse, in a subject.

In another embodiment, the present invention provides a method ofinducing formation and proliferation of a WT-1 protein-specific CTL, themethod comprising contacting a lymphocyte population with a peptide orcomposition of the present invention, thereby inducing formation andproliferation of a WT-1 protein-specific CTL. This method can beconducted in vitro, ex vivo or in vivo. When conducted in vitro or exvivo, these CTL can then be infused into a patient for therapeuticeffect.

In another embodiment, the present invention provides a method ofinducing formation and proliferation of (a) a WT-1 protein-specific CD8⁺lymphocyte; or (b) a CD4⁺ lymphocyte specific for the WT-1 protein, orthe combination thereof, the method comprising contacting a lymphocytepopulation with a peptide or composition of the present invention,thereby inducing formation and proliferation of (a) a WT-1protein-specific CD8⁺ lymphocyte; or (b) a CD4⁺ lymphocyte specific forthe WT-1 protein; or a combination thereof. This method can be conductedin vitro, ex vivo or in vivo. When conducted in vitro or ex vivo, theseCTL can then be infused into a patient for therapeutic effect.

In another embodiment, the present invention provides a method ofinducing an anti-cancer immune response in a subject, the methodcomprising the step of contacting the subject with an immunogeniccomposition comprising (a) a WT-1 protein; (b) a fragment of a WTprotein; (c) a nucleotide molecule encoding a WT-1 protein; or (d) anucleotide molecule encoding a fragment of a WT-1 protein, therebyinducing an anti-mesothelioma immune response in a subject. In oneembodiment, the fragment of a WT-1 protein consists of a peptide orcomprises a peptide from among SEQ ID NO:1-160, 162-185, 190, 191 and193. In another embodiment the fragment consists of a peptide orcomprises a peptide from among SEQ ID NO:142, 143, 144, 145, 146, 147,148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 162,163, 164, 165, 166, 167, 168, 169, 170, 171, 173, 174, 175, 176, 177,178, 179, 180, 181, 182, and 183, or SEQ ID NO:149, 156, 173, 174 and180.

In another embodiment, the present invention provides a method oftreating a subject with a cancer, the method comprising the step ofadministering to the subject an immunogenic composition comprising (a) aWT-1 protein; (b) a fragment of a WT protein; (c) a nucleotide moleculeencoding a WT-1 protein; or (d) a nucleotide molecule encoding afragment of a WT-1 protein, thereby treating a subject with amesothelioma. In one embodiment, the fragment of a WT-1 protein is apeptide from among SEQ ID NO:1-160, 162-185, 190, 191 or 193. In anotherembodiment the fragment consists of a peptide or comprises a peptidefrom among SEQ ID NO:142, 143, 144, 145, 146, 147, 148, 149, 150, 151,152, 153, 154, 155, 156, 157, 158, 159, 160, 162, 163, 164, 165, 166,167, 168, 169, 170, 171, 173, 174, 175, 176, 177, 178, 179, 180, 181,182, and 183, or SEQ ID NO:149, 156, 173, 174 and 180.

In another embodiment, the present invention provides a method ofreducing an incidence of a cancer, or its relapse, in a subject, themethod comprising the step of administering to the subject animmunogenic composition comprising (a) a WT-1 protein; (b) a fragment ofa WT protein; (c) a nucleotide molecule encoding a WT-1 protein; or (d)a nucleotide molecule encoding a fragment of a WT-1 protein, therebyreducing an incidence of a mesothelioma, or its relapse, in a subject.In one embodiment, the fragment of a WT-1protein is a peptide from amongSEQ ID NO:1-160, 162-185, 190, 191 or 193. In another embodiment thefragment consists of a peptide or comprises a peptide from among SEQ IDNO:142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155,156, 157, 158, 159, 160, 162, 163, 164, 165, 166, 167, 168, 169, 170,171, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, and 183, or SEQID NO:149, 156, 173, 174 and 180.

In another embodiment, the cancer is a WT-1-expressing cancer. In oneembodiment, the WT-1-expressing cancer is an acute myelogenous leukemia(AML). In another embodiment, the WT-1-expressing cancer is associatedwith a myelodysplastic syndrome (MDS). In another embodiment, theWT-1-expressing cancer is an MDS. In another embodiment, theWT-1-expressing cancer is a non-small cell lung cancer (NSCLC). Inanother embodiment, the WT-1-expressing cancer is a Wilms' tumor. Inanother embodiment, the WT-1-expressing cancer is a leukemia. In anotherembodiment, the WT-1-expressing cancer is a hematological cancer. Inanother embodiment, the WT-1-expressing cancer is a lymphoma. In anotherembodiment, the WT-1-expressing cancer is a desmoplastic small roundcell tumor. In another embodiment, the WT-1-expressing cancer is amesothelioma. In another embodiment, the WT-1-expressing cancer is amalignant mesothelioma. In another embodiment, the WT-1-expressingcancer is a gastric cancer. In another embodiment, the WT-1-expressingcancer is a colon cancer. In another embodiment, the WT-1-expressingcancer is a lung cancer. In another embodiment, the WT-1-expressingcancer is abreast cancer. In another embodiment, the WT-1-expressingcancer is a germ cell tumor. In another embodiment, the WT-1-expressingcancer is an ovarian cancer. In another embodiment, the WT-1-expressingcancer is a uterine cancer. In another embodiment, the WT-1-expressingcancer is a thyroid cancer. In another embodiment, the WT-1-expressingcancer is a hepatocellular carcinoma. In another embodiment, theWT-1-expressing cancer is a thyroid cancer. In another embodiment, theWT-1-expressing cancer is a liver cancer. In another embodiment, theWT-1-expressing cancer is a renal cancer. In another embodiment, theWT-1-expressing cancer is a Kaposi's sarcoma. In another embodiment, theWT-1-expressing cancer is a sarcoma. In another embodiment,theWT-1-expressing cancer is any other carcinoma or sarcoma.

In another embodiment, the WT-1-expressing cancer is a solid tumor. Inanother embodiment, the solid tumor is associated with a WT-1-expressingcancer. In another embodiment, the solid tumor is associated with amyelodysplastic syndrome (MDS). In another embodiment, the solid tumoris associated with a non-small cell lung cancer (NSCLC). In anotherembodiment, the solid tumor is associated with a lung cancer. In anotherembodiment, the solid tumor is associated with a breast cancer. Inanother embodiment, the solid tumor is associated with a colorectalcancer. In another embodiment, the solid tumor is associated with aprostate cancer. In another embodiment, the solid tumor is associatedwith an ovarian cancer. In another embodiment, the solid tumor isassociated with a renal cancer. In another embodiment, the solid tumoris associated with a pancreatic cancer. In another embodiment, the solidtumor is associated with a brain cancer. In another embodiment, thesolid tumor is associated with a gastrointestinal cancer. In anotherembodiment, the solid tumor is associated with a skin cancer. In anotherembodiment, the solid tumor is associated with a melanoma.

In another embodiment, the present invention provides a compositioncomprising an isolated peptide of the invention in combination with atleast 1 additional WT-1 peptide. In certain embodiments, a compositioncomprising at least 2 different isolated peptides of the presentinvention is provided. In certain embodiments, a composition comprisingat least 3 or at least 4 different isolated peptides of the presentinvention is provided. Each possibility represents a separate embodimentof the present invention. In certain embodiments, the composition of thepresent invention is a vaccine.

In another embodiment, the present invention provides a method oftreating a subject with a WT-1-expressing cancer, the method comprisingadministering to the subject a peptide or composition of the presentinvention, thereby treating a subject with a WT-1-expressing cancer.

In another embodiment, the present invention provides a method ofreducing the incidence of a WT-1-expressing cancer, or its relapse, in asubject, the method comprising administering to the subject a peptide orcomposition of the present invention, thereby reducing the incidence ofa WT-1-expressing cancer, or its relapse, in a subject.

In another embodiment, the present invention provides a method ofinducing formation and proliferation of a WT-1 protein-specific CTL, themethod comprising contacting a lymphocyte population with a peptide orcomposition of the present invention, thereby inducing formation andproliferation of a WT-1 protein-specific CTL.

In another embodiment, the present invention provides a method ofinducing formation and proliferation of (a) a WT-1 protein-specific CD8⁺lymphocyte; or (b) a CD4⁺ lymphocyte specific for the WT-1 protein, orthe combination thereof, the method comprising contacting a lymphocytepopulation with a peptide or composition of the present invention,thereby inducing formation and proliferation of (a) a WT-1protein-specific CD8⁺ lymphocyte; or (b) a CD4⁺ lymphocyte specific forthe WT-1 protein; or a combination thereof.

In another embodiment, the invention is directed to a peptide of theinvention with at least one amino acid change that increases theaffinity of the peptide for binding to a HLA molecule.

This application claims priority to U.S. provisional application Ser.No. 61/586,177, filed Jan. 13, 2012; and Ser. No. 61/647,207, filed May15, 2012; both of which are incorporated herein by reference in theirentireties.

BRIEF DESCRIPTION OF THE FIGURES

So that the matter in which the above-recited features, advantages andobjects of the invention, as well as others which will become clear, areattained and can be understood in detail, more particular descriptionsof the invention are briefly summarized. Details of the above may be hadby reference to certain embodiments thereof, which are illustrated inthe appended drawings. These drawings form a part of the specification.It is to be noted; however, that the appended drawings illustratepreferred embodiments of the invention and therefore are not to beconsidered limiting in their scope.

FIG. 1 A-D shows WT-1 specific responses of CTL generated from PBMC ofnormal donors (n=56) by stimulation with autologous APCs loaded withtotal pool of WT-1 derived pentadeca peptides;

FIG. 2 A-E depicts the strategy for the generation of the total pool ofoverlapping pentadeca peptides spanning the whole sequence of the WT-1protein and epitope mapping;

FIG. 3 A-D shows that the combined HLA class I and II restricted WT-1specific T cell response to the same immunodominant peptide sequencederived from WT-1 protein in the WT-1 CTL after 40 days of co-culturewith the WT-1 total pool of overlapping 15-mers loaded on autologousCAMs;

FIG. 4A-F depicts schema of WT-1; and

FIG. 5 depicts results using mixed A0201 epitopes loaded on A0201-AAPCin 8 normal A0201+ donors.

DETAILED DESCRIPTION OF THE INVENTION

This invention provides immunogenic peptides, and compositions andvaccines comprising immunogenic peptides, and methods of treating,reducing the incidence of, and inducing immune responses to aWT-1-expressing cancer, comprising administering one or more immunogenicpeptides.

This invention provides WT-1 peptides and methods of treating, reducingthe incidence of, and inducing immune responses against aWT-1-expressing cancer, comprising immunogenic peptides.

The WT-1 molecule from which the peptides of the present invention arederived has, in another embodiment, the sequence:

(SEQ ID NO: 194)   1 SRQRPHPGAL RNPTACPLPH FPPSLPPTHS PTHPPRAGTA    AQAPGPRRLL  51 AAILDFLLLQ DPASTCVPEP ASQHTLRSGP GCLQQPEQQG    VRDPGGIWAK 101 LGAAEASAER LQGRRSRGAS GSEPQQMGSD VRDLNALLPA    VPSLGGGGGC 151 ALPVSGAAQW APVLDFAPPG ASAYGSLGGP APPPAPPPPP    PPPPHSFIKQ 201 EPSWGGAEPH EEQCLSAFTV HFSGQFTGTA GACRYGPFGP    PPPSQASSGQ 251 ARMFPNAPYL PSCLESQPAI RNQGYSTVTF DGTPSYGHTP    SHHAAQFPNH 301 SFKHEDPMGQ QGSLGEQQYS VPPPVYGCHT PTDSCTGSQA    LLLRTPYSSD 351 NLYQMTSQLE CMTWNQMNLG ATLKGVAAGS SSSVKWTEGQ    SNHSTGYESD 401 NHTTPILCGA QYRIHTHGVF RGIQDVRRVP GVAPTLVRSA    SETSEKRPFM 451 CAYPGCNKRY FKLSHLQMHS RKHTGEKPYQ CDFKDCERRF    SRSDQLKRHQ 501 RRHTGVKPFQ CKTCQRKFSR SDHLKTHTRT HTGKTSEKPF    SCRWPSCQKK 551 FARSDELVRH HNMHQRNMTK LQLALThe foregoing sequence of the WT-1 protein is that published by Gessleret al. (37) which comprises 575 aminoacids and includes the first 126aminoacids in the N-terminus missing in the (Exon 5+, KTS+) isoform ofWT-116.

In another embodiment, the WT-1 sequence is

MGSDVRDLNALLPAVPSLGGGGGCALPVSGAAQWAPVLDFAPPGASAYGS LGGPAPPPAPPPPPPPPPHSFIKQEPSWGGAEPHEEQCLSAFTVHFSGQFTGTAGACRYGP FGPPPPSQASSGQARMFPNAPYLPSCLESQPAIRNQGYSTVTFDGTPSYGHTPSHHAAQFPNHS FKHEDPMGQQGSLGEQQYSVPPPVYGCHTPTDSCTGSQALLLRTPYSSDNLYQMTSQLECMT WNQMNLGATLKGVAAGSSSSVKWTEGQSNHSTGYESDNHTTPILCGAQYRIHTHGVFRGIQ DVRRVPGVAPTLVRSASETSEKRPFMCAYPGCNKRYFKLSHLQMHSRKHTGEKPYQCDFKDC ERRFSRSDQLKRHQRRHTGVKPFQCKTCQRKFSRSDHLKTHTRTHTGKTSEKPFSCRWPSCQKKFARSDELVRHHNMHQRNMTKLQLAL(GenBank Accession number AY245105; SEQ ID NO: 195).

In another embodiment, the WT-1 molecule has the sequence:

AAEASAERLQGRRSRGASGSEPQQMGSDVRDLNALLPAVPSLGGGGGCAL PVSGAAQWAPVLDFAPPGASAYGSLGGPAPPPAPPPPPPPPPHSFIKQEPSWGGAEPHEE QCLSAFTVHFSGQFTGTAGACRYGPFGPPPPSQASSGQARMFPNAPYLPSCLESQPAIRNQGYS TVTFDGTPSYGHTPSHHAAQFPNHSFKHEDPMGQQGSLGEQQYSVPPPVYGCHTPTDSCTGSQ ALLLRTPYSSDNLYQMTSQLECMTWNQMNLGATLKGHSTGYESDNHTTPILCGAQYRIHTHG VFRGIQDVRRVPGVAPTLVRSASETSEKRPFMCAYPGCNKRYFKLSHLQMHSRKHTGEKPY QCDFKDCERRFSRSDQLKRHQRRHTGVKPFQCKTCQRKFSRSDHLKTHTRTHTGEKPFSCRWPSCQKKFARS DELVRHHNMHQRNMTKLQLAL(GenBank Accession number NM_000378; SEQ ID NO: 196).

In another embodiment, the WT-1 molecule has the sequence:

MQDPASTCVPEPASQHTLRSGPGCLQQPEQQGVRDPGGIWAKLGAAEASA ERLQGRRSRGASGSEPQQMGSDVRDLNALLPAVPSLGGGGGCALPVSGAAQWAPVLDFAPP GASAYGSLGGPAPPPAPPPPPPPPPHSFIKQEPSWGGAEPHEEQCLSAFTVHFSGQFTGTA GACRYGPFGPPPPSQASSGQARMFPNAPYLPSCLESQPAIRNQGYSTVTFDGTPSYGHTPSHHAA QFPNHSFKHEDPMGQQGSLGEQQYSVPPPVYGCHTPTDSCTGSQALLLRTPYSSDNLYQMTS QLECMTWNQMNLGATLKGVAAGSSSSVKWTEGQSNHSTGYESDNHTTPILCGAQYRIHTH GVFRGIQDVRRVPGVAPTLVRSASETSEKRPFMCAYPGCNKRYFKLSHLQMHSRKHTGEKPY QCDFKDCERRFSRSDQLKRHQRRHTGVKPFQCKTCQRKFSRSDHLKTHTRTHTGEKPFSCRWPSCQKKFARS DELVRHHNMHQRNMTKLQLAL(GenBank Accession number NP_077742; SEQ ID No: 197).

In another embodiment, the WT-1 protein has the sequence set forth inGenBank Accession # NM_(—)024426. In other embodiments, the WT-1 proteinhas or comprises one of the sequences set forth in one of the followingsequence entries: NM_(—)024425, NM_(—)024424, NM_(—)000378, 595530,D13624, D12496, D 12497, or X77549. In another embodiment, the WT-1protein has any other WT-1 sequence known in the art. This inventionprovides peptides, compositions, and immunogenic compositions such asvaccines comprising immunogenic peptides, and methods of treating,reducing the incidence of, and inducing immune responses to aWT-1-expressing cancer, comprising administering immunogenic peptides.

In one embodiment, the present invention provides an isolated WT-1peptide having an amino acid (AA) sequence consisting of any one of thesequences SEQ ID NO:1-160, 162-185, 190, 191 and 193. In one embodiment,the present invention provides an isolated HLA class I binding WT-1peptide having an amino acid (AA) sequence consisting of any one of thesequences SEQ ID NO:142, 143, 144, 145, 146, 147, 148, 149, 150, 151,152, 153, 154, 155, 156, 157, 158, 159, 160, 162, 163, 164, 165, 166,167, 168, 169, 170, 171, 173, 174, 175, 176, 177, 178, 179, 180, 181,182, and 183. In one embodiment, the present invention provides anisolated HLA class II binding WT-1 peptide having an amino acid (AA)sequence consisting of any one of the sequences SEQ ID NO:149, 156, 173,174 and 180. In another embodiment the HLA class I peptides consist ofor comprise SEQ ID NO:142, 143, 144, 145, 146, 147, 148, 150 or 151, andthe HLA class II peptide consists of or comprises SEQ ID NO:149.

In one embodiment, the present invention provides an isolated WT-1peptide having an amino acid (AA) sequence comprising any one of thesequences SEQ ID NO:1-53 or 43-XXX, or a fragment thereof. In oneembodiment, the present invention provides an isolated HLA class Ibinding WT-1 peptide having an amino acid (AA) sequence comprising ofany one of the sequences SEQ ID NO:142, 143, 144, 145, 146, 147, 148,149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 162, 163,164, 165, 166, 167, 168, 169, 170, 171, 173, 174, 175, 176, 177, 178,179, 180, 181, 182, and 183. In one embodiment, the present inventionprovides an isolated HLA class II binding WT-1 peptide having an aminoacid (AA) sequence comprising of any one of the sequences SEQ ID NO:149,156, 173, 174 and 180. In another embodiment the HLA class I peptidesconsist of or comprise SEQ ID NO:142, 143, 144, 145, 146, 147, 148, 150or 151, and the HLA class II peptide consists of or comprises SEQ IDNO:149.

In another embodiment, the present invention provides a compositioncomprising (a) an antigen-presenting cell and (b) a peptide selectedfrom SEQ ID NO:1-160, 162-185, 190, 191 and 193. In another embodiment,the present invention provides a composition comprising (a) anantigen-presenting cell and (b) an HLA class I binding peptide selectedfrom SEQ ID NO:142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152,153, 154, 155, 156, 157, 158, 159, 160, 162, 163, 164, 165, 166, 167,168, 169, 170, 171, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182,and 183. In another embodiment, the present invention provides acomposition comprising (a) an antigen-presenting cell and (b) an HLAclass II binding peptide selected from SEQ ID NO:149, 156, 173, 174 and180. In another embodiment the HLA class I peptides consist of orcomprise SEQ ID NO:142, 143, 144, 145, 146, 147, 148, 150 or 151, andthe HLA class II peptide consists of or comprises SEQ ID NO:149.

In another embodiment, the present invention provides a vaccinecomprising one or more peptides of SEQ ID NO:1-160, 162-185, 190, 191and 193. In another embodiment, the present invention provides a vaccinecomprising one or more HLA class I binding peptides selected from SEQ IDNO:142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155,156, 157, 158, 159, 160, 162, 163, 164, 165, 166, 167, 168, 169, 170,171, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, and 183. Inanother embodiment, the present invention provides a vaccine comprisingone or more HLA class II binding peptides selected from SEQ ID NO:149,156, 173, 174 and 180. In another embodiment, the present inventionprovides a vaccine comprising one or more HLA class I binding peptidesselected from SEQ ID NO:142, 143, 144, 145, 146, 147, 148, 149, 150,151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 162, 163, 164, 165,166, 167, 168, 169, 170, 171, 173, 174, 175, 176, 177, 178, 179, 180,181, 182, and 183, and one or more HLA class II binding peptidesselected from SEQ ID NO:149, 156, 173, 174 and 180. In anotherembodiment the HLA class I peptides consist of or comprise SEQ IDNO:142, 143, 144, 145, 146, 147, 148, 150 or 151, and the HLA class IIpeptide consists of or comprises SEQ ID NO:149.

In another embodiment, the present invention provides a method oftreating a subject with a WT-1-expressing cancer, the method comprisingadministering to the subject a WT-1peptide or vaccine of the presentinvention, thereby treating a subject with a WT-1-expressing cancer.

In another embodiment, the present invention provides a method ofreducing the incidence of a WT-1-expressing cancer, or its relapse, in asubject, the method comprising administering to the subject a WT-1peptide or vaccine of the present invention, thereby reducing theincidence of a WT-1-expressing cancer, orits relapse, in a subject.

In another embodiment, the present invention provides a method ofinducing an anti-cancer immune response in a subject, the methodcomprising the step of contacting the subject with an immunogeniccomposition comprising (a) a WT-1 protein; (b) a fragment of a WTprotein; (c) a nucleotide molecule encoding a WT-1 protein; or (d) anucleotide molecule encoding a fragment of a WT-1 protein, therebyinducing an anti-mesothelioma immune response in a subject. In oneembodiment, the fragment of a WT-1 protein consists of a peptide orcomprises a peptide from among SEQ ID NO:1-160, 162-185, 190, 191 and193. In another embodiment the fragment consists of a peptide orcomprises a peptide from among SEQ ID NO:142, 143, 144, 145, 146, 147,148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 162,163, 164, 165, 166, 167, 168, 169, 170, 171, 173, 174, 175, 176, 177,178, 179, 180, 181, 182, and 183, or SEQ ID NO:149, 156, 173, 174 and180.

In another embodiment, the present invention provides a method oftreating a subject with a cancer, the method comprising the step ofadministering to the subject an immunogenic composition comprising (a) aWT-1 protein; (b) a fragment of a WT protein; (c) a nucleotide moleculeencoding a WT-1 protein; or (d) a nucleotide molecule encoding afragment of a WT-1 protein, thereby treating a subject with amesothelioma. In one embodiment, the fragment of a WT-1 protein is apeptide from among SEQ ID NO:1-160, 162-185, 190, 191 and 193. Inanother embodiment the fragment consists of a peptide or comprises apeptide from among SEQ ID NO:142, 143, 144, 145, 146, 147, 148, 149,150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 162, 163, 164,165, 166, 167, 168, 169, 170, 171, 173, 174, 175, 176, 177, 178, 179,180, 181, 182, and 183, or SEQ ID NO:149, 156, 173, 174 and 180. Inanother embodiment the HLA class I peptides consist of or comprise SEQID NO:142, 143, 144, 145, 146, 147, 148, 150 or 151, and the HLA classII peptide consists of or comprises SEQ ID NO:149.

In another embodiment, the present invention provides a method ofreducing an incidence of a cancer, or its relapse, in a subject, themethod comprising the step of administering to the subject animmunogenic composition comprising (a) a WT-1 protein; (b) a fragment ofa WT protein; (c) a nucleotide molecule encoding a WT-1 protein; or (d)a nucleotide molecule encoding a fragment of a WT-1 protein, therebyreducing an incidence of a mesothelioma, or its relapse, in a subject.In one embodiment, the fragment of a WT-1 protein is a peptide fromamong SEQ ID NO:1-160, 162-185, 190, 191 or 193. In another embodimentthe fragment consists of a peptide or comprises a peptide from among SEQID NO:142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154,155, 156, 157, 158, 159, 160, 162, 163, 164, 165, 166, 167, 168, 169,170, 171, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, and 183, orSEQ ID NO:149, 156, 173, 174 and 180. In another embodiment the HLAclass I peptides consist of or comprise SEQ ID NO:142, 143, 144, 145,146, 147, 148, 150 or 151, and the HLA class II peptide consists of orcomprises SEQ ID NO:149.

In another embodiment, the present invention provides a method oftreating a subject with a WT-1-expressing cancer, the method comprisingadministering to the subject a WT-1peptide or vaccine of the presentinvention, thereby treating a subject with a WT-1-expressing cancer.

In another embodiment, the present invention provides a method ofreducing the incidence of a WT-1-expressing cancer, or its relapse, in asubject, the method comprising administering to the subject a WT-1peptide or vaccine of the present invention, thereby reducing theincidence of a WT-1-expressing cancer, orits relapse, in a subject.

In another embodiment, the present invention provides a method ofinducing an anti-cancer immune response in a subject, the methodcomprising the step of contacting the subject with an immunogeniccomposition comprising (a) a WT-1 protein; (b) a fragment of a WTprotein; (c) a nucleotide molecule encoding a WT-1 protein; or (d) anucleotide molecule encoding a fragment of a WT-1 protein, therebyinducing an anti-mesothelioma immune response in a subject. In oneembodiment, the fragment of a WT-1 protein is a peptide from among SEQID NO:1-160, 162-185, 190, 191 or 193. In another embodiment thefragment consists of a peptide or comprises a peptide from among SEQ IDNO:142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155,156, 157, 158, 159, 160, 162, 163, 164, 165, 166, 167, 168, 169, 170,171, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, and 183, or SEQID NO:149, 156, 173, 174 and 180. In another embodiment the HLA class Ipeptides consist of or comprise SEQ ID NO:142, 143, 144, 145, 146, 147,148, 150 or 151, and the HLA class II peptide consists of or comprisesSEQ ID NO:149.

In another embodiment, the present invention provides a method oftreating a subject with a cancer, the method comprising the step ofadministering to the subject an immunogenic composition comprising (a) aWT-1 protein; (b) a fragment of a WT protein; (c) a nucleotide moleculeencoding a WT-1 protein; or (d) a nucleotide molecule encoding afragment of a WT-1 protein, thereby treating a subject with amesothelioma. In one embodiment, the fragment of a WT-1 protein is apeptide from among SEQ ID NO:1-160, 162-185, 190, 191 or 193.

In another embodiment, the present invention provides a method ofreducing an incidence of a cancer, or its relapse, in a subject, themethod comprising the step of administering to the subject animmunogenic composition comprising (a) a WT-1 protein; (b) a fragment ofa WT protein; (c) a nucleotide molecule encoding a WT-1 protein; or (d)a nucleotide molecule encoding a fragment of a WT-1 protein, therebyreducing an incidence of a mesothelioma, or its relapse, in a subject.In one embodiment, the fragment of a WT-1 protein is a peptide fromamong SEQ ID NO:1-160, 162-185, 190, 191 or 193.

In another embodiment, the cancer is a WT-1-expressing cancer. In oneembodiment, the WT-1-expressing cancer is an acute myelogenous leukemia(AML). In another embodiment, the WT-1-expressing cancer is associatedwith a myelodysplastic syndrome (MDS). In another embodiment, theWT-1-expressing cancer is an MDS. In another embodiment, theWT-1-expressing cancer is a non-small cell lung cancer (NSCLC). Inanother embodiment, the WT-1-expressing cancer is a Wilms' tumor. Inanother embodiment, the WT-1-expressing cancer is a leukemia. In anotherembodiment, the WT-1-expressing cancer is a hematological cancer. Inanother embodiment, the WT-1-expressing cancer is a lymphoma. In anotherembodiment, the WT-1-expressing cancer is a desmoplastic small roundcell tumor. In another embodiment, the WT-1-expressing cancer is amesothelioma. In another embodiment, the WT-1-expressing cancer is amalignant mesothelioma. In another embodiment, the WT-1-expressingcancer is a gastric cancer. In another embodiment, the WT-1-expressingcancer is a colon cancer. In another embodiment, the WT-1-expressingcancer is a lung cancer. In another embodiment, the WT-1-expressingcancer is abreast cancer. In another embodiment, the WT-1-expressingcancer is a germ cell tumor. In another embodiment, the WT-1-expressingcancer is an ovarian cancer. In another embodiment, the WT-1-expressingcancer is a uterine cancer. In another embodiment, the WT-1-expressingcancer is a thyroid cancer. In another embodiment, the WT-1-expressingcancer is a hepatocellular carcinoma. In another embodiment, theWT-1-expressing cancer is a thyroid cancer. In another embodiment, theWT-1-expressing cancer is a liver cancer. In another embodiment, theWT-1-expressing cancer is a renal cancer. In another embodiment, theWT-1-expressing cancer is a Kaposi's sarcoma. In another embodiment, theWT-1-expressing cancer is a sarcoma. In another embodiment, theWT-1-expressing cancer is any other carcinoma or sarcoma.

In another embodiment, the WT-1-expressing cancer is a solid tumor. Inanother embodiment, the solid tumor is associated with a WT-1-expressingcancer. In another embodiment, the solid tumor is associated with amyelodysplastic syndrome (MDS). In another embodiment, the solid tumoris associated with a non-small cell lung cancer (NSCLC). In anotherembodiment, the solid tumor is associated with a lung cancer. In anotherembodiment, the solid tumor is associated with a breast cancer. Inanother embodiment, the solid tumor is associated with a colorectalcancer. In another embodiment, the solid tumor is associated with aprostate cancer. In another embodiment, the solid tumor is associatedwith an ovarian cancer. In another embodiment, the solid tumor isassociated with a renal cancer. In another embodiment, the solid tumoris associated with a pancreatic cancer. In another embodiment, the solidtumor is associated with a brain cancer. In another embodiment, thesolid tumor is associated with a gastrointestinal cancer. In anotherembodiment, the solid tumor is associated with a skin cancer. In anotherembodiment, the solid tumor is associated with a melanoma.

In another embodiment, the present invention provides a compositioncomprising an isolated peptide of the invention in combination with atleast 1 additional WT-1 peptide. In certain embodiments, a compositioncomprising at least 2 different isolated peptides of the presentinvention is provided. In certain embodiments, a composition comprisingat least 3 or at least 4 different isolated peptides of the presentinvention is provided. Each possibility represents a separate embodimentof the present invention. In certain embodiments, the composition of thepresent invention is a vaccine.

In another embodiment, the present invention provides a method oftreating a subject with a WT-1-expressing cancer, the method comprisingadministering to the subject a peptide or composition of the presentinvention, thereby treating a subject with a WT-1-expressing cancer.

In another embodiment, the present invention provides a method ofreducing the incidence of a WT-1-expressing cancer, or its relapse, in asubject, the method comprising administering to the subject a peptide orcomposition of the present invention, thereby reducing the incidence ofa WT-1-expressing cancer, or its relapse, in a subject.

In another embodiment, the present invention provides a method ofinducing formation and proliferation of a WT-1 protein-specific CTL, themethod comprising contacting a lymphocyte population with a peptide orcomposition of the present invention, thereby inducing formation andproliferation of a WT-1 protein-specific CTL.

In another embodiment, the present invention provides a method ofinducing formation and proliferation of (a) a WT-1 protein-specific CD8⁺lymphocyte; or (b) a CD4⁺ lymphocyte specific for the WT-1 protein, orthe combination thereof, the method comprising contacting a lymphocytepopulation with a peptide or composition of the present invention,thereby inducing formation and proliferation of (a) a WT-1protein-specific CD8⁺ lymphocyte; or (b) a CD4⁺ lymphocyte specific forthe WT-1 protein; or a combination thereof.

In another embodiment, the invention is directed to a peptide of theinvention with at least one amino acid change that increases theaffinity of the peptide for binding to a HLA molecule.

“Peptide,” in another embodiment of methods and compositions of thepresent invention, refers to a compound of subunit AA connected bypeptide bonds. In another embodiment, the peptide comprises an AAanalogue. In another embodiment, the peptide comprises a peptidomimetic.The different AA analogues and peptidomimetics that can be included inthe peptides of methods and compositions of the present invention areenumerated hereinbelow. The subunits are, in another embodiment, linkedby peptide bonds. In another embodiment, the subunit is linked byanother type of bond, e.g. ester, ether, etc. Each possibilityrepresents a separate embodiment of the present invention.

The unaltered peptides of the present invention (as described both aboveand below) are referred to collectively herein as “WT-1 peptides.” Eachof the embodiments enumerated below for “WT-1 peptides” applies tounaltered WT-1 peptides and HLA class I and class II heterocliticpeptides of the present invention. Each possibility represents aseparate embodiment of the present invention.

In another embodiment, a WT-1 peptide of the present invention binds toan HLA class I molecule or a class II molecule. In another embodimentthe peptide binds to both a class I and a class II molecule. In anotherembodiment, the HLA class II molecule is an HLA-DRB molecule. In anotherembodiment, the HLA class I1-molecule is an HLA-DRA molecule. In anotherembodiment, the HLA molecule is an HLA-DQA1 molecule. In anotherembodiment, the HLA molecule is an HLA-DQB1 molecule. In anotherembodiment, the HLA molecule is an HLA-DPA1 molecule. In anotherembodiment, the HLA molecule is an HLA-DPB 1 molecule. In anotherembodiment, the HLA molecule is an HLA-DMA molecule. In anotherembodiment, the HLA molecule is an HLA-DMB molecule. In anotherembodiment, the HLA molecule is an HLA-DOA molecule. In anotherembodiment, the HLA molecule is an HLA-DOB molecule. In anotherembodiment, the HLA molecule is any other HLA class I1-molecule known inthe art. Each possibility represents a separate embodiment of thepresent invention.

In another embodiment, the HLA class I molecule whose binding motif iscontained in or comprising a peptide of the present invention is, inanother embodiment, an HLA-A molecule. In another embodiment, the HLAclass I molecule is an HLA-B molecule. In another embodiment, the HLAclass I molecule is an HLA-C molecule. In another embodiment, the HLAclass I molecule is an HLA-A0201 molecule. In another embodiment, themolecule is HLA A1. In another embodiment, the HLA class I molecule isHLA A2. In another embodiment, the HLA class I molecule is HLA A2.1. Inanother embodiment, the HLA class I molecule is HLA A3. In anotherembodiment, the HLA class I molecule is HLA A3.2. In another embodiment,the HLA class I molecule is HLA All. In another embodiment, the HLAclass I molecule is HLA A24. In another embodiment, the HLA class Imolecule is HLA B7. In another embodiment, the HLA class I molecule isHLA B27. In another embodiment, the HLA class I molecule is HLA B8. Eachpossibility represents a separate embodiment of the present invention.

In another embodiment, the HLA class I molecule-binding WT-1 peptide ofmethods and compositions of the present invention binds to a superfamilyof HLA class I molecules. In another embodiment, the superfamily is theA2 superfamily. In another embodiment, the superfamily is the A3superfamily. In another embodiment, the superfamily is the A24superfamily. In another embodiment, the superfamily is the B7superfamily. In another embodiment, the superfamily is the B27superfamily. In another embodiment, the superfamily is the B44superfamily. In another embodiment, the superfamily is the C1superfamily. In another embodiment, the superfamily is the C4superfamily. In another embodiment, the superfamily is any othersuperfamily known in the art. Each possibility represents a separateembodiment of the present invention.

In another embodiment, the HLA molecule is a A0101, A0201, A0203, A2402,A6901, B0702, A3101, B3501, B3503, B3508, B3802, B3801, B3901, B4001,B4402, B4701, B5701, C0401, C1701, DRB₁0101, DRB₁0402, DRB₁0402,DRB₁0401 or DRB₁1104 molecule. In another embodiment, the peptides ofSEQ ID NO:142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153,154, 155, 156, 157, 158, 159, 160, 162, 163, 164, 165, 166, 167, 168,169, 170, 171, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, and183, and SEQ ID NO:149, 156, 173, 174 and 180, bind to the HLA class Ior class II molecules described for each peptide in Tables 1 or 2. Inanother embodiment the HLA class I peptides consist of or comprise SEQID NO:142, 143, 144, 145, 146, 147, 148, 150 or 151, and the HLA classII peptide consists of or comprises SEQ ID NO:149, and bind to thecorresponding HLA molecule or molecules indicated for each peptide inTable 1 or Table 2. In one embodiment, certain peptides can bind to morethan one HLA allele.

In another embodiment, a modification of a peptide of the invention isprovided. In one embodiment the modification comprises at least oneheteroclitic amino acid change, also referred to as a mutation ormutated, or an anchor residue mutation (see below). An HLA class Imolecule binding motif of a modified peptide of the present inventionexhibits an increased affinity for the HLA class I molecule, relative tothe unmutated counterpart of the peptide. In another embodiment, thepoint mutation increases the affinity of the isolated, mutated WT-1peptide for the HLA class I molecule. In another embodiment, theincrease in affinity is relative to the affinity (for the same HLA classI molecule) of the isolated, unmutated WT-1 peptide wherefrom theisolated, mutated WT-1 peptide was derived. Each possibility representsa separate embodiment of the present invention.

In another embodiment, a WT-1 peptide of methods and compositions of thepresent invention is so designed as to exhibit affinity for an HLAmolecule. In another embodiment, the affinity is a high affinity, asdescribed herein.

HLA molecules, known in another embodiment as major histocompatibilitycomplex (MHC) molecules, bind peptides and present them to immune cells.Thus, in another embodiment, the immunogenicity of a peptide ispartially determined by its affinity for HLA molecules. HLA class Imolecules interact with CD8 molecules, which are generally present oncytotoxic T lymphocytes (CTL). HLA class II molecules interact with CD4molecules, which are generally present on helper T lymphocytes.

In another embodiment, a peptide of the present invention isimmunogenic. In another embodiment, “immunogenic” refers to an abilityto stimulate, elicit or participate in an immune response. In anotherembodiment, the immune response elicited is a cell-mediated immuneresponse. In another embodiment, the immune response is a combination ofcell-mediated and humoral responses.

In another embodiment, T cells that bind to the MHC molecule-peptidecomplex become activated and induced to proliferate and lyse cellsexpressing a protein comprising the peptide. T cells are typicallyinitially activated by “professional” antigen presenting cells (“APC”;e.g. dendritic cells, monocytes, and macrophages), which presentcostimulatory molecules that encourage T cell activation as opposed toanergy or apoptosis. In another embodiment, the response isheteroclitic, as described herein, such that the CTL lyses a neoplasticcell expressing a protein which has an AA sequence homologous to apeptide of this invention, or a different peptide than that used tofirst stimulate the T cell.

In another embodiment, an encounter of a T cell with a peptide of thisinvention induces its differentiation into an effector and/or memory Tcell. Subsequent encounters between the effector or memory T cell andthe same peptide, or, in another embodiment, with a related peptide ofthis invention, leads to a faster and more intense immune response. Suchresponses are gauged, in another embodiment, by measuring the degree ofproliferation of the T cell population exposed to the peptide. Inanother embodiment, such responses are gauged by any of the methodsenumerated hereinbelow.

In another embodiment, the peptides of methods and compositions of thepresent invention bind an HLA class II molecule with high affinity. Inother embodiments, the HLA class II molecule is any HLA class IImolecule enumerated herein. Each possibility represents a separateembodiment of the present invention.

In another embodiment, derivatives of peptides of methods andcompositions of the present invention bind an HLA class I molecule withhigh affinity. In other embodiments, the MHC class I molecule is any MHCclass I molecule enumerated herein. Each possibility represents aseparate embodiment of the present invention.

In another embodiment, a peptide of methods and compositions of thepresent invention binds an HLA class II molecule with significantaffinity, while a peptide derived from the original peptide binds an HLAclass I molecule with significant affinity.

In another embodiment, “affinity” refers to the concentration of peptidenecessary for inhibiting binding of a standard peptide to the indicatedMHC molecule by 50%. In another embodiment, “high affinity” refers to anaffinity is such that a concentration of about 500 nanomolar (nM) orless of the peptide is required for 50% inhibition of binding of astandard peptide. In another embodiment, a concentration of about 400 nMor less of the peptide is required. In another embodiment, the bindingaffinity is 300 nM. In another embodiment, the binding affinity is 200nM. In another embodiment, the binding affinity is 150 nM. In anotherembodiment, the binding affinity is 100 nM. In another embodiment, thebinding affinity is 80 nM. In another embodiment, the binding affinityis 60 nM. In another embodiment, the binding affinity is 40 nM. Inanother embodiment, the binding affinity is 30 nM. In anotherembodiment, the binding affinity is 20 nM. In another embodiment, thebinding affinity is 15 nM. In another embodiment, the binding affinityis 10 nM. In another embodiment, the binding affinity is 8 nM. Inanother embodiment, the binding affinity is 6 nM. In another embodiment,the binding affinity is 4 nM. In another embodiment, the bindingaffinity is 3 nM. In another embodiment, the binding affinity is 2 nM.In another embodiment, the binding affinity is 1.5 nM. In anotherembodiment, the binding affinity is 1 nM. In another embodiment, thebinding affinity is 0.8 nM. In another embodiment, the binding affinityis 0.6 nM. In another embodiment, the binding affinity is 0.5 nM. Inanother embodiment, the binding affinity is 0.4 nM. In anotherembodiment, the binding affinity is 0.3 nM. In another embodiment, thebinding affinity is less than 0.3 nM.

In another embodiment, “affinity” refers to a measure of bindingstrength to the MHC molecule. In another embodiment, affinity ismeasured using a method known in the art to measure competitive bindingaffinities. In another embodiment, affinity is measured using a methodknown in the art to measure relative binding affinities. In anotherembodiment, the method is a competitive binding assay. In anotherembodiment, the method is radioimmunoas say or RIA. In anotherembodiment, the method is BiaCore analyses. In another embodiment, themethod is any other method known in the art. In another embodiment, themethod yields an IC50 in relation to an IC50 of a reference peptide ofknown affinity.

Each type of affinity and method of measuring affinity represents aseparate embodiment of the present invention.

In another embodiment, “high affinity” refers to an IC50 of 0.5-500 nM.In another embodiment, the IC50 is 1-300 nM. In another embodiment, theIC50 is 1.5-200 nM. In another embodiment, the IC50 is 2-100 nM. Inanother embodiment, the IC50 is 3-100 nM. In another embodiment, theIC50 is 4-100 nM. In another embodiment, the IC50 is 6-100 nM. Inanother embodiment, the IC50 is 10-100 nM. In another embodiment, theIC50 is 30-100 nM. In another embodiment, the IC50 is 3-80 nM. Inanother embodiment, the IC50 is 4-60 nM. In another embodiment, the IC50is 5-50 nM. In another embodiment, the IC50 is 6-50 nM. In anotherembodiment, the IC50 is 8-50 nM. In another embodiment, the IC50 is10-50 nM. In another embodiment, the IC50 is 20-50 nM. In anotherembodiment, the IC50 is 6-40 nM. In another embodiment, the IC50 is 8-30nM. In another embodiment, the IC50 is 10-25 nM. In another embodiment,the IC50 is 15-25 nM. Each affinity and range of affinities represents aseparate embodiment of the present invention.

In another embodiment, a peptide of methods and compositions of thepresent invention binds to a superfamily of HLA molecules. Superfamiliesof HLA molecules share very similar or identical binding motifs. Inanother embodiment, the superfamily is a HLA class I superfamily. Inanother embodiment, the superfamily is a HLA class II superfamily. Eachpossibility represents a separate embodiment of the present invention.

The terms “HLA-binding peptide,” “HLA class I molecule-binding peptide,”and “HLA class II molecule-binding peptide” refer, in anotherembodiment, to a peptide that binds an HLA molecule with measurableaffinity. In another embodiment, the terms refer to a peptide that bindsan HLA molecule with high affinity. In another embodiment, the termsrefer to a peptide that binds an HLA molecule with sufficient affinityto activate a T cell precursor. In another embodiment, the terms referto a peptide that binds an HLA molecule with sufficient affinity tomediate recognition by a T cell. The HLA molecule is, in otherembodiments, any of the HLA molecules enumerated herein. Eachpossibility represents a separate embodiment of the present invention.

“Heteroclitic” refers, in another embodiment, to a peptide thatgenerates an immune response that recognizes the original peptide fromwhich the heteroclitic peptide was derived (e.g. the peptide notcontaining the anchor residue mutations). In another embodiment,“original peptide” refers to a peptide of the present invention. Inanother embodiment, “heteroclitic” refers to a peptide that generates animmune response that recognizes the original peptide from which theheteroclitic peptide was derived, wherein the immune response generatedby vaccination with the heteroclitic peptide is greater than the immuneresponse generated by vaccination with the original peptide. In anotherembodiment, a “heteroclitic” immune response refers to an immuneresponse that recognizes the original peptide from which the improvedpeptide was derived (e.g. the peptide not containing the anchor residuemutations). In another embodiment, a “heteroclitic” immune responserefers to an immune response that recognizes the original peptide fromwhich the heteroclitic peptide was derived, wherein the magnitude of theimmune response generated by vaccination with the heteroclitic peptideis greater than the immune response generated by vaccination with theoriginal peptide. In another embodiment, the magnitude of the immuneresponse generated by vaccination with the heteroclitic peptide isgreater than the immune response substantially equal to the response tovaccination with the original peptide. In another embodiment, themagnitude of the immune response generated by vaccination with theheteroclitic peptide is greater than the immune response less than theresponse to vaccination with the original peptide. In anotherembodiment, a heteroclitic peptide of the present invention is an HLAclass I heteroclitic peptide. Methods for identifying HLA class I andclass II residues, and for improving HLA binding by mutating theresidues, are well known in the art, as described below. Eachpossibility represents a separate embodiment of the present invention.

In another embodiment, a heteroclitic peptide of the present inventioninduces an immune response that is increased at least 2-fold relative tothe WT-1 peptide from which the heteroclitic peptide was derived(“native peptide”). In another embodiment, the increase is 3-foldrelative to the native peptide. In another embodiment, the increase is5-fold relative to the native peptide. In another embodiment, theincrease is 7-fold relative to the native peptide. In anotherembodiment, the increase is 10-fold relative to the native peptide. Inanother embodiment, the increase is 15-fold relative to the nativepeptide. In another embodiment, the increase is 20-fold relative to thenative peptide. In another embodiment, the increase is 30-fold relativeto the native peptide. In another embodiment, the increase is 50-foldrelative to the native peptide. In another embodiment, the increase is100-fold relative to the native peptide. In another embodiment, theincrease is 150-fold relative to the native peptide. In anotherembodiment, the increase is 200-fold relative to the native peptide. Inanother embodiment, the increase is 300-fold relative to the nativepeptide. In another embodiment, the increase is 500-fold relative to thenative peptide. In another embodiment, the increase is 1000-foldrelative to the native peptide. In another embodiment, the increase ismore than 1000-fold relative to the native peptide. Each possibilityrepresents a separate embodiment of the present invention.

In another embodiment, the present invention provides a HLA class IIheteroclitic peptide derived from an isolated WT-1 peptide of thepresent invention. In another embodiment, the process of derivingcomprises introducing a mutation that enhances a binding of the peptideto an HLA class II molecule. In another embodiment, the process ofderiving consists of introducing a mutation that enhances a binding ofthe peptide to an HLA class I molecule. In another embodiment, themutation is in an HLA class II anchor residue. In another embodiment, aheteroclitic class II peptide of the present invention is identified andtested in a manner analogous to identification and testing of HLA classI heteroclitic peptides, as exemplified herein. Each possibilityrepresents a separate embodiment of the present invention.

In another embodiment, the HLA class II binding site in a peptide of thepresent invention is created or improved by mutation of an HLA class IImotif anchor residue. In another embodiment, the anchor residue that ismodified is in the P1 position. In another embodiment, the anchorresidue is at the P2 position. In another embodiment, the anchor residueis at the P6 position. In another embodiment, the anchor residue is atthe P9 position. In another embodiment, the anchor residue is selectedfrom the P1, P2, P6, and P9 positions. In another embodiment, the anchorresidue is at the P3 position. In another embodiment, the anchor residueis at the P4 position. In another embodiment, the anchor residue is atthe P5 position. In another embodiment, the anchor residue is at the P6position. In another embodiment, the anchor residue is at the P8position. In another embodiment, the anchor residue is at the P10position. In another embodiment, the anchor residue is at the P1 1position. In another embodiment, the anchor residue is at the P12position. In another embodiment, the anchor residue is at the P13position. In another embodiment, the anchor residue is at any otheranchor residue of an HLA class II molecule that is known in the art. Inanother embodiment, residues other than P1, P2, P6, and P9 serve assecondary anchor residues; therefore, mutating them can improve HLAclass II binding. Each possibility represents a separate embodiment ofthe present invention.

In another embodiment, a heteroclitic peptide is generated byintroduction of a mutation that creates an anchor motif. “Anchor motifs”or “anchor residues” refers, in another embodiment, to 1 or a set ofpreferred residues at particular positions in an HLA-binding sequence.In another embodiment, the HLA-binding sequence is an HLA classII-binding sequence. In another embodiment, the HLA-binding sequence isan HLA class I-binding sequence. In another embodiment, the positionscorresponding to the anchor motifs are those that play a significantrole in binding the HLA molecule. In another embodiment, the anchorresidue is a primary anchor motif. In another embodiment, the anchorresidue is a secondary anchor motif. Each possibility represents aseparate embodiment of the present invention.

Methods for predicting MHC class H epitopes are well known in the art.In another embodiment, the MHC class II epitope is predicted usingTEPITOPE (Meister G E, Roberts C G et al, Vaccine 1995 13: 581-91). Inanother embodiment, the MHC class II epitope is predicted usingEpiMatrix (De Groot A S, Jesdale B M et al, AIDS Res Hum Retroviruses1997 13: 529-31). In another embodiment, the MHC class II epitope ispredicted using the Predict Method (Yu K, Petrovsky N et al, Mol Med.2002 8: 137-48). In another embodiment, the MHC class II epitope ispredicted using the SYFPEITHI epitope prediction algorithm (Examples).In another embodiment, the MHC class II epitope is predicted usingRankpep. In another embodiment, the MHC class II epitope is predictedusing any other method known in the art. Each possibility represents aseparate embodiment of the present invention.

In another embodiment, in the case of HLA class II-binding peptides(e.g. HLA-DR-binding peptides), the anchor residue that is modified isin the P1 position. In another embodiment, the anchor residue is in theP2 position. In another embodiment, the anchor residue is in the P6position. In another embodiment, the anchor residue is in the P9position. In other embodiments, the anchor residue is the P3, P4, P5,P6, P8, P10, P11, P12, or P13 position. In another embodiment, theanchor residue is any other anchor residue of an HLA class II moleculethat is known in the art. In another embodiment, residues other than P1,P2, P6, and P9 serve as secondary anchor residues; therefore, mutatingthem can improve HLA class II binding. In another embodiment, anycombination of the above residues is mutated. Each possibilityrepresents a separate embodiment of the present invention.

In another embodiment, a WT-1 peptide of the present invention binds to2 distinct HLA class II molecules. In another embodiment, the peptidebinds to three distinct HLA class II molecules. In another embodiment,the peptide binds to four distinct HLA class II molecules. In anotherembodiment, the peptide binds to five distinct HLA class II molecules.In another embodiment, the peptide binds to six distinct HLA class IImolecules. In another embodiment, the peptide binds to more than sixdistinct HLA class II molecules.

In another embodiment, the HLA class II molecules that are bound by aWT-1 peptide of the present invention are encoded by two or moredistinct alleles at a given HLA class II locus. In another embodiment,the HLA class II molecules are encoded by 3 distinct alleles at a locus.In another embodiment, the HLA class II molecules are encoded by 4distinct alleles at a locus. In another embodiment, the HLA class IImolecules are encoded by 5 distinct alleles at a locus. In anotherembodiment, the HLA class II molecules are encoded by 6 distinct allelesat a locus. In another embodiment, the HLA class II molecules areencoded by more than six distinct alleles at a locus.

In another embodiment, the HLA class II molecules bound by the WT-1peptide are encoded by HLA class II genes at 2 distinct loci. In anotherembodiment, the HLA molecules bound are encoded by HLA class II genes at2 or more distinct loci. In another embodiment, the HLA molecules boundare encoded by HLA class II genes at 3 distinct loci. In anotherembodiment, the HLA molecules bound are encoded by HLA class II genes at3 or more distinct loci. In another embodiment, the HLA molecules boundare encoded by HLA class II genes at 4 distinct loci. In anotherembodiment, the HLA molecules bound are encoded by HLA class II genes at4 or more distinct loci. In another embodiment, the HLA molecules boundare encoded by HLA class II genes at more than 4 distinct loci. In otherembodiments, the loci are selected from HLA-DRB loci. In anotherembodiment, the HLA class II-binding peptide is an HLA-DRA bindingpeptide. In another embodiment, the peptide is an HLA-DQA1 bindingpeptide. In another embodiment, the peptide is an HLA-DQB 1 bindingpeptide. In another embodiment, the peptide is an HLA-DPA1 bindingpeptide. In another embodiment, the peptide is an HLA-DPB 1 bindingpeptide. In another embodiment, the peptide is an HLA-DMA bindingpeptide. In another embodiment, the peptide is an HLA-DMB bindingpeptide. In another embodiment, the peptide is an HLA-DOA bindingpeptide. In another embodiment, the peptide is an HLA-DOB bindingpeptide. In another embodiment, the peptide binds to any other HLA classII molecule known in the art. Each possibility represents a separateembodiment of the present invention.

In another embodiment, a WT-1 peptide of the present invention binds to2 distinct HLA-DRB molecules. In another embodiment, the peptide bindsto 3 distinct HLA-DRB molecules. In another embodiment, the peptidebinds to 4 distinct HLA-DRB molecules. In another embodiment, thepeptide binds to 5 distinct HLA-DRB molecules. In another embodiment,the peptide binds to 6 distinct HLA-DRB molecules. In anotherembodiment, the peptide binds to more than 6 distinct HLA-DRB molecules.

In another embodiment, a WT-1 peptide of the present invention binds toHLA-DRB molecules that are encoded by 2 distinct HLA-DRB alleles. Inanother embodiment, the HLA-DRB molecules are encoded by 3 distinctHLA-DRB alleles. In another embodiment, the HLA-DRB molecules areencoded by 4 distinct HLA-DRB alleles. In another embodiment, theHLA-DRB molecules are encoded by 5 distinct HLA-DRB alleles. In anotherembodiment, the HLA-DRB molecules are encoded by 6 distinct HLA-DRBalleles. In another embodiment, the HLA-DRB molecules are encoded bymore than 6 distinct HLA-DRB alleles. Each possibility represents aseparate embodiment of the present invention.

In another embodiment, a WT-1 peptide of the present invention binds toHLA-DRB molecules that are encoded by 2 distinct HLA-DRB allelesselected from DRB 101, DRB 301, DRB 401, DRB 701, DRB 1101, and DRB1501. In another embodiment, the WT-1 peptide binds to HLA-DRB moleculesencoded by 3 distinct HLA-DRB alleles selected from DRB 101, DRB 301,DRB 401, DRB 701, DRB 1101, and DRB 1501. In another embodiment, theWT-1 peptide binds to HLA-DRB molecules encoded by 4 distinct HLA-DRBalleles selected from DRB 101, DRB 301, DRB 401, DRB 701, DRB 1101, andDRB 1501. In another embodiment, the WT-1 peptide binds to HLA-DRBmolecules encoded by 5 distinct HLA-DRB alleles selected from DRB 101,DRB 301, DRB 401, DRB 701, DRB 1101, DRB 1104 and DRB 1501. In anotherembodiment, the WT-1 peptide binds to HLA-DRB molecules encoded by eachof the following HLA-DRB alleles: DRB 101, DRB 301, DRB 401, DRB 701,DRB 1101, and DRB 1501. Each possibility represents a separateembodiment of the present invention.

In another embodiment, the present invention provides a compositioncomprising 2 distinct WT-1 peptides of the present invention. In anotherembodiment, the 2 distinct WT-1 peptides are both unaltered. In anotherembodiment, 1 of the WT-1 peptides is unaltered, while the other isheteroclitic. In another embodiment, both of the WT-1 peptides areheteroclitic.

In another embodiment, the composition comprises 3 distinct WT-1peptides of the present invention. In another embodiment, thecomposition comprises 4 distinct WT-1 peptides of the present invention.In another embodiment, the composition comprises 5 distinct WT-1peptides of the present invention. In another embodiment, thecomposition comprises more than 5 distinct isolated WT-1 peptides of thepresent invention.

In another embodiment, 2 of the WT-1 peptides in the composition areunaltered. In another embodiment, 2 of the WT-1 peptides in thecomposition are heteroclitic. In another embodiment, 2 of the WT-1peptides in the composition are unaltered, and 2 are heteroclitic. Inanother embodiment, more than 2 of the WT-1 peptides in the compositionare unaltered. In another embodiment, more than 2 of the WT-1 peptidesin the composition are heteroclitic.

In another embodiment, more than 2 of the WT-1 peptides in thecomposition are unaltered, and more than 2 are heteroclitic. Eachpossibility represents a separate embodiment of the present invention.

In another embodiment, 1 of the additional WT-1 peptides in acomposition of the present invention has a sequence selected from thesequences set forth in SEQ ID No: 1-160, 162-185, 190, 191 or 193. Inanother embodiment, 2 of the additional WT-1 peptides have a sequenceselected from the sequences set forth in SEQ ID No: 1-160, 162-185, 190,191 or 193. In another embodiment, 3 of the additional WT-1 peptideshave a sequence selected from the sequences set forth in SEQ ID No:1-160, 162-185, 190, 191 or 193.

In another embodiment, any other immunogenic WT-1 peptide known in theart is utilized as an additional WT-1 peptide. In another embodiment,any combination of immunogenic WT-1 peptides known in the art isutilized. Non-limiting sources of other WT peptides includeWO2005053618, WO2007047764 and WO2007120673.

Each additional WT-1 peptide, and each combination thereof, represents aseparate embodiment of the present invention.

In another embodiment, a composition of the present invention contains 2HLA class II heteroclitic peptides that are derived from the sameisolated WT-1 peptide of the present invention. In another embodiment,the 2 HLA class II heteroclitic peptides contain mutations in differentHLA class II molecule anchor residues. In another embodiment, the 2 HLAclass II heteroclitic peptides contain different mutations in the sameanchor residues. In another embodiment, 2 of the HLA class IIheteroclitic peptides are derived from different isolated WT-1 peptidesof the present invention. Each possibility represents a separateembodiment of the present invention.

In another embodiment, 2 WT-1 peptides of the present invention, or theWT-1 peptides that correspond to two HLA class II heteroclitic peptidesof the present invention, overlap with one another. In anotherembodiment, the overlap between the peptides is at least 7 amino acids(AA). In another embodiment, the overlap is at least 8 AA. In anotherembodiment, the overlap is at least 9 AA. In another embodiment, theoverlap is 7 AA. In another embodiment, the overlap is 8 AA. In anotherembodiment, the overlap is 9 AA. In another embodiment, the overlap is10 AA. In another embodiment, the overlap is 11 AA. In anotherembodiment, the overlap is 12 AA. In another embodiment, the overlap is13 AA. In another embodiment, the overlap is 14 AA. In anotherembodiment, the overlap is 15 AA. In another embodiment, the overlap is16 AA. In another embodiment, the overlap is more than 16 AA. Eachpossibility represents a separate embodiment of the present invention.

In another embodiment, the peptides in a composition of the presentinvention bind to 2 distinct HLA class II molecules. In anotherembodiment, the peptides bind to 3 distinct HLA class II molecules. Inanother embodiment, the peptides bind to 4 distinct HLA class IImolecules. In another embodiment, the peptides bind to 5 distinct HLAclass II molecules. In another embodiment, the peptides bind to morethan 5 distinct HLA class II molecules. In another embodiment, thepeptides in the composition bind to the same HLA class II molecules.

In another embodiment, each of the WT 1 peptides in a composition of thepresent invention binds to a set of HLA class II molecules. In anotherembodiment, each of the WT-1 peptides binds to a distinct set of HLAclass II molecules. In another embodiment, the WT-1 peptides in thecomposition bind to the same set of HLA class II molecules. In anotherembodiment, 2 of the WT-1 peptides bind to a distinct but overlappingset of HLA class II molecules. In another embodiment, 2 or more of theWT-1 peptides bind to the same set of HLA class II molecules, whileanother of the WT-1 peptides binds to a distinct set. In anotherembodiment, 2 or more of the WT-1 peptides bind to an overlapping set ofHLA class II molecules, while another of the WT-1 peptides binds to adistinct set.

In another embodiment, 2 or more of the WT-1 peptides in a compositionof the present invention each binds to more than 1 HLA-DRB molecule. Inanother embodiment, the 4 or more HLA-DRB molecules bound by thepeptides in the composition are distinct from one another. In anotherembodiment, the HLA-DRB molecules are encoded by different HLA-DRBalleles. Each possibility represents a separate embodiment of thepresent invention.

In another embodiment, 2 or more of the HLA class II molecules bound byWT-1 peptides in a composition of the present invention are HLA-DRBmolecules. In another embodiment, 3 or more of the HLA class IImolecules that are bound are HLA-DRB molecules. In other embodiments,the HLA class II molecules that are bound can be any of the HLA class IImolecules enumerated herein. In another embodiment, the HLA class IImolecules that are bound are encoded by 2 or more distinct HLA class IIalleles at a given locus. In another embodiment, the HLA class IImolecules that are bound are encoded by HLA class II genes at 2 or moredistinct loci.

Each of the above compositions represents a separate embodiment of thepresent invention.

In another embodiment, a “set of HLA class II molecules” refers to theHLA class II molecules encoded by different alleles at a particularlocus. In another embodiment, the term refers to HLA class II moleculeswith a particular binding specificity. In another embodiment, the termrefers to HLA class II molecules with a particular peptide consensussequence. In another embodiment, the term refers to a superfamily of HLAclass II molecules. Each possibility represents a separate embodiment ofthe present invention.

In another embodiment, the present invention provides a compositioncomprising an unaltered HLA class II molecule-binding WT-1 peptide ofthe present invention and a second, HLA class I molecule-binding WT-1peptide. In another embodiment, the composition comprises more than 1HLA class II molecule-binding WT-1 peptide of the present invention, inaddition to the HLA class I molecule-binding WT-1 peptide. In anotherembodiment, the composition comprises more than 1 HLA class Imolecule-binding WT-1 peptide, in addition to the HLA class IImolecule-binding WT-1 peptide. Each possibility represents a separateembodiment of the present invention.

In another embodiment, the AA sequence of the HLA class Imolecule-binding WT-1 peptide comprises a sequence selected from SEQ IDNo: 1-160, 162-185, 190, 191 or 193. In another embodiment, the AAsequence of the HLA class I molecule-binding WT-1 peptide is selectedfrom the sequences set forth in SEQ ID No: 1-160, 162-185, 190, 191 or193. Each possibility represents a separate embodiment of the presentinvention.

In another embodiment, the HLA class I molecule-binding WT-1 peptide isan HLA class I heteroclitic peptide. In another embodiment, the HLAclass I molecule-binding WT-1 peptide contains a mutation in an HLAclass I molecule anchor residue thereof, as described further herein. Asprovided herein, WT-1-derived peptides were modified in HLA anchorresidues to generate heteroclitic peptides with increased predictedbinding to HLA-A0201 and HLA-A0301. Peptides with increased predictedbinding also exhibited enhanced ability to bind HLA class I moleculesand increased immunogenicity.

In another embodiment, the mutation that enhances MHC binding is in theresidue at position 1 of the HLA class I heteroclitic peptide. Inanother embodiment, the residue is changed to tyrosine. In anotherembodiment, the residue is changed to glycine. In another embodiment,the residue is changed to threonine. In another embodiment, the residueis changed to phenylalanine. In another embodiment, the residue ischanged to any other residue known in the art. In another embodiment, asubstitution in position 1 (e.g. to tyrosine) stabilizes the binding ofthe position 2 anchor residue.

In another embodiment, the mutation is in position 2 of the HLA class Iheteroclitic peptide. In another embodiment, the residue is changed toleucine. In another embodiment, the residue is changed to valine. Inanother embodiment, the residue is changed to isoleucine. In anotherembodiment, the residue is changed to methionine. In another embodiment,the residue is changed to any other residue known in the art.

In another embodiment, the mutation is in position 6 of the HLA class Iheteroclitic peptide. In another embodiment, the residue is changed tovaline. In another embodiment, the residue is changed to cysteine. Inanother embodiment, the residue is changed to glutamine. In anotherembodiment, the residue is changed to histidine. In another embodiment,the residue is changed to any other residue known in the art.

In another embodiment, the mutation is in position 9 of the HLA class Iheteroclitic peptide. In another embodiment, the mutation changes theresidue at the C-terminal position thereof. In another embodiment, theresidue is changed to valine. In another embodiment, the residue ischanged to threonine. In another embodiment, the residue is changed toisoleucine. In another embodiment, the residue is changed to leucine. Inanother embodiment, the residue is changed to alanine. In anotherembodiment, the residue is changed to cysteine. In another embodiment,the residue is changed to any other residue known in the art.

In another embodiment, the point mutation is in a primary anchorresidue. In another embodiment, the HLA class I primary anchor residuesare positions 2 and 9. In another embodiment, the point mutation is in asecondary anchor residue. In another embodiment, the HLA class Isecondary anchor residues are positions 1 and 8. In another embodiment,the HLA class I secondary anchor residues are positions 1, 3, 6, 7, and8. In another embodiment, the point mutation is in a position selectedfrom positions 4, 5, and 8. Each possibility represents a separateembodiment of the present invention.

In another embodiment, the point mutation is in 1 or more residues inpositions selected from positions 1, 2, 8, and 9 of the HLA class Ibinding motif. In another embodiment, the point mutation is in 1 or moreresidues in positions selected from positions 1, 3, 6, and 9. In anotherembodiment, the point mutation is in 1 or more residues in positionsselected from positions 1, 2, 6, and 9. In another embodiment, the pointmutation is in 1 or more residues in positions selected from positions1, 6, and 9. In another embodiment, the point mutation is in 1 or moreresidues in positions selected from positions 1, 2, and 9. In anotherembodiment, the point mutation is in 1 or more residues in positionsselected from positions 1, 3, and 9. In another embodiment, the pointmutation is in 1 or more residues in positions selected from positions 2and 9. In another embodiment, the point mutation is in 1 or moreresidues in positions selected from positions 6 and 9. Each possibilityrepresents a separate embodiment of the present invention.

Each of the above anchor residues and substitutions represents aseparate embodiment of the present invention.

In another embodiment, the HLA class I molecule-binding WT peptide haslength of 9 AA. In another embodiment, the peptide has length of 10 AA.As provided herein, native and heteroclitic peptides of 9-10 AAexhibited substantial binding to HLA class I molecules and ability toelicit cytokine secretion and cytolysis by CTL.

In another embodiment, the HLA class I molecule that is bound by the HLAclass I molecule-binding WT-1 peptide is an HLA-A molecule. In anotherembodiment, the HLA class I-molecule is an HLA-A2 molecule. In anotherembodiment, the HLA class I-molecule is an HLA-A3 molecule. In anotherembodiment, the HLA class I-molecule is an HLA-Al 1 molecule. In anotherembodiment, the HLA class I-molecule is an HLA-B 8 molecule. In anotherembodiment, the HLA class I-molecule is an HLA-0201 molecule. In anotherembodiment, the HLA class I-molecule binds any other HLA class Imolecule known in the art. Each possibility represents a separateembodiment of the present invention.

In another embodiment, a WT-1 peptide of methods and compositions of thepresent invention has a length of 8-30 amino acids. In anotherembodiment, the peptide has a length of 9-11 AA. In another embodiment,the peptide ranges in size from 7-25 AA, or in another embodiment, 8-11,or in another embodiment, 8-15, or in another embodiment, 9-20, or inanother embodiment, 9-18, or in another embodiment, 9-15, or in anotherembodiment, 8-12, or in another embodiment, 9-11 AA in length. Inanother embodiment, the peptide is 8 AA in length, or in anotherembodiment, 9 AA or in another embodiment, 10 AA or in anotherembodiment, 12 AA or in another embodiment, 25 AA in length, or inanother embodiment, any length therebetween. In another embodiment, thepeptide is of greater length, for example 50, or 100, or more. In thisembodiment, the cell processes the peptide to a length of 7 and 25 AA inlength. In this embodiment, the cell processes the peptide to a lengthof 9-11 AA Each possibility represents a separate embodiment of thepresent invention.

In another embodiment, the peptide is 15-23 AA in length. In anotherembodiment, the length is 15-24 AA. In another embodiment, the length is15-25 AA. In another embodiment, the length is 15-26 AA. In anotherembodiment, the length is 15-27 AA. In another embodiment, the length is15-28 AA. In another embodiment, the length is 14-30 AA. In anotherembodiment, the length is 14-29 AA. In another embodiment, the length is14-28 AA. In another embodiment, the length is 14-26 AA. In anotherembodiment, the length is 14-24 AA. In another embodiment, the length is14-22 AA. In another embodiment, the length is 14-20 AA. In anotherembodiment, the length is 16-30 AA. In another embodiment, the length is16-28 AA. In another embodiment, the length is 16-26 AA. In anotherembodiment, the length is 16-24 AA. In another embodiment, the length is16-22 AA. In another embodiment, the length is 18-30 AA. In anotherembodiment, the length is 18-28 AA. In another embodiment, the length is18-26 AA. In another embodiment, the length is 18-24 AA. In anotherembodiment, the length is 18-22 AA. In another embodiment, the length is18-20 AA. In another embodiment, the length is 20-30 AA. In anotherembodiment, the length is 20-28 AA. In another embodiment, the length is20-26 AA. In another embodiment, the length is 20-24 AA. In anotherembodiment, the length is 22-30 AA. In another embodiment, the length is22-28 AA. In another embodiment, the length is 22-26 AA. In anotherembodiment, the length is 24-30 AA. In another embodiment, the length is24-28 AA. In another embodiment, the length is 24-26 AA.

Each of the above peptides, peptide lengths, and types of peptidesrepresents a separate embodiment of the present invention.

In another embodiment, minor modifications are made to peptides of thepresent invention without decreasing their affinity for HLA molecules orchanging their TCR specificity, utilizing principles well known in theart. In the case of HLA class I-binding peptides, “minor modifications”refers, in another embodiment, to e.g. insertion, deletion, orsubstitution of one AA, inclusive, or deletion or addition of 1-3 AAoutside of the residues between 2 and 9, inclusive. While the computeralgorithms described herein are useful for predicting the MHC classI-binding potential of peptides, they have 60-80% predictive accuracy;and thus, the peptides should be evaluated empirically before a finaldetermination of MHC class I-binding affinity is made. Thus, peptides ofthe present invention are not limited to peptides predicated by thealgorithms to exhibit strong MHC class I-binding affinity. The types aremodifications that can be made are listed below. Each modificationrepresents a separate embodiment of the present invention.

In another embodiment, a peptide enumerated in the Examples of thepresent invention is further modified by mutating an anchor residue toan MHC class I preferred anchor residue, which can be, in otherembodiments, any of the anchor residues enumerated herein. In anotherembodiment, a peptide of the present invention containing an MHC class Ipreferred anchor residue is further modified by mutating the anchorresidue to a different MHC class I preferred residue for that location.The different preferred residue can be, in other embodiments, any of thepreferred residues enumerated herein.

In another embodiment, the anchor residue that is further modified is inthe 1 position. In another embodiment, the anchor residue is in the 2position. In another embodiment, the anchor residue is in the 3position. In another embodiment, the anchor residue is in the 4position. In another embodiment, the anchor residue is in the 5position. In another embodiment, the anchor residue is in the 6position. In another embodiment, the anchor residue is in the 7position. In another embodiment, the anchor residue is in the 8position. In another embodiment, the anchor residue is in the 9position. In the case of HLA class I-binding peptides, residues otherthan 2 and 9 can serve as secondary anchor residues; therefore, mutatingthem can improve MHC class I binding. Each possibility represents aseparate embodiment of the present invention.

In another embodiment, a peptide of methods and compositions of thepresent invention is a length variant of a peptide enumerated in theExamples. In another embodiment, the length variant is one amino acid(AA) shorter than the peptide from the Examples. In another embodiment,the length variant is two AA shorter than the peptide from the Examples.In another embodiment, the length variant is more than two AA shorterthan the peptide from the Examples. In another embodiment, the shorterpeptide is truncated on the N-terminal end. In another embodiment, theshorter peptide is truncated on the C-terminal end. In anotherembodiment, the truncated peptide is truncated on both the N-terminaland C-terminal ends. Peptides are, in another embodiment, amenable totruncation without changing affinity for HLA molecules, as is well knownin the art.

Each of the above truncated peptides represents a separate embodiment ofthe present invention.

In another embodiment, the length variant is longer than a peptideenumerated in the Examples of the present invention. In anotherembodiment, the longer peptide is extended on the N-terminal end inaccordance with the surrounding WT-1 sequence. Peptides are, in anotherembodiment, amenable to extension on the N-terminal end without changingaffinity for HLA molecules, as is well known in the art. Such peptidesare thus equivalents of the peptides enumerated in the Examples. Inanother embodiment, the N-terminal extended peptide is extended by oneresidue. In another embodiment, the N-terminal extended peptide isextended by two residues. In another embodiment, the N-terminal extendedpeptide is extended by three residues. In another embodiment, theN-terminal extended peptide is extended by more than three residues.

In another embodiment, the longer peptide is extended on the C terminalend in accordance with the surrounding WT-1 sequence. Peptides are, inanother embodiment, amenable to extension on the C-terminal end withoutchanging affinity for HLA molecules, as is well known in the art. Suchpeptides are thus equivalents of the peptides enumerated in the Examplesof the present invention. In another embodiment, the C-terminal extendedpeptide is extended by one residue. In another embodiment, theC-terminal extended peptide is extended by two residues. In anotherembodiment, the C-terminal extended peptide is extended by threeresidues. In another embodiment, the C-terminal extended peptide isextended by more than three residues.

In another embodiment, the extended peptide is extended on both theN-terminal and C-terminal ends in accordance with the surrounding WT-1sequence.

Each of the above extended peptides represents a separate embodiment ofthe present invention.

In another embodiment, a truncated peptide of the present inventionretains the HLA anchor residues (e.g. the HLA class I anchor residues)on the second residue and the C-terminal residue, with a smaller numberof intervening residues (e.g. 5) than a peptide enumerated in theExamples of the present invention. Peptides are, in another embodiment,amenable to such mutation without changing affinity for

HLA molecules. In another embodiment, such a truncated peptide isdesigned by removing one of the intervening residues of one of the abovesequences. In another embodiment, the HLA anchor residues are retainedon the second and eighth residues. In another embodiment, the HLA anchorresidues are retained on the first and eighth residues. Each possibilityrepresents a separate embodiment of the present invention.

In another embodiment, an extended peptide of the present inventionretains the HLA anchor residues (e.g. the HLA class I anchor residues)on the second residue and the C-terminal residue, with a larger numberof intervening residues (e.g. 7 or 8) than a peptide enumerated in theExamples of the present invention. In another embodiment, such anextended peptide is designed by adding one or more residues between twoof the intervening residues of one of the above sequences. It is wellknown in the art that residues can be removed from or added between theintervening sequences of HLA-binding peptides without changing affinityfor HLA. Such peptides are thus equivalents of the peptides enumeratedin the Examples of the present invention. In another embodiment, the HLAanchor residues are retained on the second and ninth residues. Inanother embodiment, the HLA anchor residues are retained on the firstand eighth residues. In another embodiment, the HLA anchor residues areretained on the two residues separated by six intervening residues. Eachpossibility represents a separate embodiment of the present invention.

“Fragment,” in another embodiment, refers to a peptide of 11 or more AAin length. In another embodiment, a peptide fragment of the presentinvention is 16 or more AA long. In another embodiment, the fragment is12 or more AA long. In another embodiment, the fragment is 13 or moreAA. In another embodiment, the fragment is 14 or more AA. In anotherembodiment, the fragment is 15 or more AA. In another embodiment, thefragment is 17 or more AA. In another embodiment, the fragment is 18 ormore AA. In another embodiment, the fragment is 19 or more AA. Inanother embodiment, the fragment is 22 or more AA. In anotherembodiment, the fragment is 8-12 AA. In another embodiment, the fragmentis about 8-12 AA. In another embodiment, the fragment is 16-19 AA. Inanother embodiment, the fragment is about 16-19 AA. In anotherembodiment, the fragment 10-25 AA. In another embodiment, the fragmentis about 10-25 AA. In another embodiment, the fragment has any otherlength. Each possibility represents a separate embodiment of the presentinvention.

“Fragment of a WT-1 protein,” in another embodiment, refers to any ofthe definitions of “fragment” found herein. Each definition represents aseparate embodiment of the present invention.

In another embodiment, a peptide of the present invention is homologousto a peptide enumerated in the Examples. The terms “homology,”“homologous,” etc., when in reference to any protein or peptide, refer,in another embodiment, to a percentage of amino acid residues in thecandidate sequence that are identical with the residues of acorresponding native polypeptide, after aligning the sequences andintroducing gaps, if necessary, to achieve the maximum percent homology,and not considering any conservative substitutions as part of thesequence identity. Methods and computer programs for the alignment arewell known in the art.

In another embodiment, the term “homology,” when in reference to anynucleic acid sequence similarly indicates a percentage of nucleotides ina candidate sequence that are identical with the nucleotides of acorresponding native nucleic acid sequence.

Homology is, in another embodiment, determined by computer algorithm forsequence alignment, by methods well described in the art. In otherembodiments, computer algorithm analysis of nucleic acid sequencehomology includes the utilization of any number of software packagesavailable, such as, for example, the BLAST, DOMAIN, BEAUTY (BLASTEnhanced Alignment Utility), GENPEPT and TREMBL packages.

In another embodiment, “homology” refers to identity to a sequenceselected from SEQ ID No: 1-160, 162-185, 190, 191 or 193 of greater than70%. In another embodiment, “homology” refers to identity to a sequenceselected from SEQ ID No: 1-160, 162-185, 190, 191 or 193 of greater than72%. In another embodiment, “homology” refers to identity to one of SEQID No: 1-160, 162-185, 190, 191 or 193 of greater than 75%. In anotherembodiment, “homology” refers to identity to a sequence selected fromSEQ ID No: 1-160, 162-185, 190, 191 or 193 of greater than 78%. Inanother embodiment, “homology” refers to identity to one of SEQ ID No:1-160, 162-185, 190, 191 or 193 of greater than 80%. In anotherembodiment, “homology” refers to identity to one of SEQ ID No: 1-160,162-185, 190, 191 or 193 of greater than 82%. In another embodiment,“homology” refers to identity to a sequence selected from SEQ ID No:1-160, 162-185, 190, 191 or 193 of greater than 83%. In anotherembodiment, “homology” refers to identity to one of SEQ ID No: 1-160,162-185, 190, 191 or 193 of greater than 85%. In another embodiment,“homology” refers to identity to one of SEQ ID No: 1-160, 162-185, 190,191 or 193 of greater than 87%. In another embodiment, “homology” refersto identity to a sequence selected from SEQ ID No: 1-160, 162-185, 190,191 or 193 of greater than [0128] 88%. In another embodiment, “homology”refers to identity to one of SEQ ID No: 1-160, 162-185, 190, 191 or 193of greater than 90%. In another embodiment, “homology” refers toidentity to one of SEQ ID No: 1-160, 162-185, 190, 191 or 193 of greaterthan 92%. In another embodiment, “homology” refers to identity to asequence selected from SEQ ID No: 1-160, 162-185, 190, 191 or 193 ofgreater than 93%. In another embodiment, “homology” refers to identityto one of SEQ ID No: 1-160, 162-185, 190, 191 or 193 of greater than95%. In another embodiment, “homology” refers to identity to a sequenceselected from SEQ ID No: 1-160, 162-185, 190, 191 or 193 of greater than96%. In another embodiment, “homology” refers to identity to one of SEQID No: 1-160, 162-185, 190, 191 or 193 of greater than 97%. In anotherembodiment, “homology” refers to identity to one of SEQ ID No: 1-160,162-185, 190, 191 or 193 of greater than 98%. In another embodiment,“homology” refers to identity to one of SEQ ID No: 1-160, 162-185, 190,191 or 193 of greater than 99%. In another embodiment, “homology” refersto identity to one of SEQ ID No: 1-160, 162-185, 190, 191 or 193 of100%. Each possibility represents a separate embodiment of the presentinvention. [00114] In another embodiment, homology is determined viadetermination of candidate sequence hybridization, methods of which arewell described in the art (See, for example, “Nucleic AcidHybridization”Hames, B. D., and Higgins S. J., Eds. (1985); Sambrook et al., 2001,Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Press, N. Y.;and Ausubel et al., 1989, Current Protocols in Molecular Biology, GreenPublishing Associates and Wiley Interscience, N. Y). In anotherembodiments, methods of hybridization are carried out under moderate tostringent conditions, to the complement of a DNA encoding a nativecaspase peptide. Hybridization conditions being, for example, overnightincubation at 42° C. in a solution comprising: 10-20% formamide, 5×SSC(150 mM NaCl, 15 mM trisodium citrate), 50 mM sodium phosphate (pH 7.6),5×Denhardt's solution, 10% dextran sulfate, and 20 μg/ml denatured,sheared salmon sperm DNA.

Each of the above homologues and variants of peptides enumerated in theExamples represents a separate embodiment of the present invention.

In another embodiment, the present invention provides a compositioncomprising a peptide of this invention. In another embodiment, thecomposition further comprises a pharmaceutically acceptable carrier. Inanother embodiment, the composition further comprises an adjuvant. Inanother embodiment, the composition comprises 2 or more peptides of thepresent invention. In another embodiment, the composition furthercomprises any of the additives, compounds, or excipients set forthhereinbelow. In another embodiment, the adjuvantis KLH, QS21, Freund'scomplete or incomplete adjuvant, aluminum phosphate, aluminum hydroxide,BCG or alum. In other embodiments, the carrier is any carrier enumeratedherein. In other embodiments, the adjuvant is any adjuvant enumeratedherein. Each possibility represents a separate embodiment of the presentinvention.

In another embodiment, this invention provides a vaccine comprising apeptide of this invention. In another embodiment, this inventionprovides a vaccine comprising an antigen-presenting cell (APC) and apeptide of this invention. In another embodiment, the vaccine furthercomprises a carrier. In another embodiment, the vaccine furthercomprises an adjuvant. In another embodiment, the vaccine furthercomprises an APC. In another embodiment, the vaccine further comprises acombination of more than 1 of an antigen, carrier, and/or APC. Inanother embodiment, the vaccine is a cell-based composition. Eachpossibility represents a separate embodiment of the present invention.

In another embodiment, the term “vaccine” refers to a material orcomposition that, when introduced into a subject, provides aprophylactic or therapeutic response for a particular disease,condition, or symptom of same. In another embodiment, this inventioncomprises peptide-based vaccines, wherein the peptide comprises anyembodiment listed herein, including immunomodulating compounds such ascytokines, adjuvants, etc.

In another embodiment, a vaccine of methods and compositions of thepresent invention further comprises an adjuvant. In another embodiment,the adjuvant is a Montanide. In another embodiment the adjuvant isMontanide ISA 51. Montanide ISA 51 contains a natural metabolizable oiland a refined emulsifier. In another embodiment, the adjuvant is GM-CSF.Recombinant GM-CSF is a human protein grown, in another embodiment, in ayeast (S. cerevisiae) vector. GM-CSF promotes clonal expansion anddifferentiation of hematopoietic progenitor cells, APC, and dendriticcells and T cells.

In another embodiment, the adjuvant is a cytokine. In anotherembodiment, the adjuvant is a growth factor. In another embodiment, theadjuvant is a cell population. In another embodiment, the adjuvant isQS21. In another embodiment, the adjuvant is Freund's incompleteadjuvant. In another embodiment, the adjuvant is aluminum phosphate. Inanother embodiment, the adjuvant is aluminum hydroxide. In anotherembodiment, the adjuvant is BCG. In another embodiment, the adjuvant isalum.

In another embodiment, the adjuvant is an interleukin. In anotherembodiment, the adjuvant is a chemokine. In another embodiment, theadjuvant is any other type of adjuvant known in the art. In anotherembodiment, the WT-1 vaccine comprises two the above adjuvants. Inanother embodiment, theWT-1 vaccine comprises more than two the aboveadjuvants. Each possibility represents a separate embodiment of thepresent invention.

In other embodiments, a vaccine or composition of the present inventioncan comprise any of the embodiments of WT-1 peptides of the presentinvention and combinations thereof. Each possibility represents aseparate embodiment of the present invention.

It is to be understood that any embodiments described herein, regardingpeptides, vaccines and compositions of this invention can be employed inany of the methods of this invention. Each combination of peptide,vaccine, or composition with a method represents an embodiment thereof.

In another embodiment, the present invention provides a method oftreating a subject with a WT-1-expressing cancer, the method comprisingadministering to the subject a WT-1 vaccine of the present invention,thereby treating a subject with a WT-1-expressing cancer.

In another embodiment, the present invention provides a method oftreating a subject with an MDS, the method comprising administering tothe subject a WT-1 vaccine of the present invention, thereby treating asubject with an MDS.

In another embodiment, the present invention provides a method ofsuppressing or halting the progression of a WT-1-expressing cancer in asubject, the method comprising administering to the subject a WT-1vaccine of the present invention, thereby suppressing or halting theprogression of a WT-1-expressing cancer.

In another embodiment, the present invention provides a method ofreducing the incidence of a WT-1-expressing cancer in a subject, themethod comprising administering to the subject a WT-1 vaccine of thepresent invention, thereby reducing the incidence of a WT-1-expressingcancer in a subject.

In another embodiment, the present invention provides a method ofreducing the incidence of an AML in a subject, the method comprisingadministering to the subject a WT-1 vaccine of the present invention,thereby reducing the incidence of an AML.

In another embodiment, the present invention provides a method ofreducing the incidence of relapse of a WT-1-expressing cancer in asubject, the method comprising administering to the subject a WT-1vaccine of the present invention, thereby reducing the incidence ofrelapse of a WT-1-expressing cancer in a subject.

In another embodiment, the present invention provides a method ofreducing the incidence of relapse of an AML in a subject, the methodcomprising administering to the subject a WT-1 vaccine of the presentinvention, thereby reducing the incidence of relapse of an AML in asubject.

In another embodiment, the present invention provides a method ofbreaking a T cell tolerance of a subject to a WT-1-expressing cancer,the method comprising administering to the subject a WT-1 vaccine of thepresent invention, thereby breaking a T cell tolerance to aWT-1-expressing cancer.

In another embodiment, the present invention provides a method oftreating a subject having aWT-1-expressing cancer, comprising (a)inducing in a donor formation and proliferation of human cytotoxic Tlymphocytes (CTL) that recognize a malignant cell of the cancer by amethod of the present invention; and (b) infusing the human CTL into thesubject, thereby treating a subject having a cancer.

In another embodiment, the present invention provides a method oftreating a subject having a WT 1-expressing cancer, comprising (a)inducing ex vivo formation and proliferation of human CTL that recognizea malignant cell of the cancer by a method of the present invention,wherein the human immune cells are obtained from a donor; and (b)infusing the human CTL into the subject, thereby treating a subjecthaving a cancer.

In another embodiment, the present invention provides a method ofinducing formation and proliferation of (a) a WT-1 protein-specific CD8⁺lymphocyte; or (b) a CD4⁺ lymphocyte specific for the WT-1 protein, orthe combination thereof, the method comprising contacting a lymphocytepopulation with a peptide or composition of the present invention,thereby inducing formation and proliferation of (a) a WT-1protein-specific CD8⁺ lymphocyte; or (b) a CD4⁺ lymphocyte specific forthe WT-1 protein; or a combination thereof. This method can be conductedin vitro, ex vivo or in vivo. When conducted in vitro or ex vivo, theseCTL can then be infused into a patient for therapeutic effect.

Methods for ex vivo immunotherapy are well known in the art and aredescribed, for example, in United States Patent Application SerialNumbers 2006/0057130, 2005/0221481, 2005/0214268, 2003/0175272,2002/0127718, and U.S. Pat. No. 5,229,115, which are incorporated hereinby reference. Additional methods are well known in the art and aredescribed, for example, in Davis I D et al (Blood dendritic cellsgenerated with Flt3 ligand and CD40 ligand prime CD8+ T cellsefficiently in cancer patients. J Immunother. 2006 September-October;29(5):499-511) and Mitchell M S et al (The cytotoxic T cell response topeptide analogs of the HLA-A*0201-restricted MUC1 signal sequenceepitope, M1.2. Cancer Immunol Immunother. 2006 Jul. 28). Each methodrepresents a separate embodiment of the present invention.

In another embodiment, the present invention provides a method ofinducing the formation and proliferation of CTL specific for cells of aWT-1-expressing cancer, the method comprising contacting a lymphocytepopulation with a vaccine of the present invention. In anotherembodiment, the vaccine is an APC associated with a peptide of thepresent invention. In another embodiment, the vaccine is an APCassociated with a mixture of peptides of the present invention. Eachpossibility represents a separate embodiment of the present invention.

In another embodiment, this invention provides a method of generating aheteroclitic immune response in a subject, wherein the heterocliticimmune response is directed against a WT-1-expressing cancer, the methodcomprising administering to the subject a vaccine of the presentinvention, thereby generating a heteroclitic immune response.

In another embodiment, the present invention provides a method ofinducing an anti-mesothelioma immune response in a subject, the methodcomprising the step of contacting the subject with an immunogeniccomposition comprising (a) a WT-1 protein; or (b) a fragment of a WTprotein, thereby inducing an anti-mesothelioma immune response in asubject. In another embodiment, the mesothelioma is a malignantmesothelioma. Each possibility represents a separate embodiment of thepresent invention.

In another embodiment, the present invention provides a method ofinducing an anti-mesothelioma immune response in a subject, the methodcomprising the step of contacting the subject with an immunogeniccomposition comprising a nucleotide molecule encoding (a) a WT-1protein; or (b) a fragment of a WT-1 protein, thereby inducing ananti-mesothelioma immune response in a subject. In another embodiment,the mesothelioma is a malignant mesothelioma. Each possibilityrepresents a separate embodiment of the present invention.

In another embodiment, the present invention provides a method oftreating a subject with a mesothelioma, the method comprising the stepof administering to the subject an immunogenic composition comprising(a) a WT-1 protein; or (b) a fragment of a WT protein, thereby treatinga subject with a mesothelioma. In another embodiment, the mesotheliomais a malignant mesothelioma. Each possibility represents a separateembodiment of the present invention.

In another embodiment, the present invention provides a method oftreating a subject with a mesothelioma, the method comprising the stepof administering to the subject an immunogenic composition comprising anucleotide molecule encoding (a) a WT-1 protein; or (b) a fragment of aWT-1 protein, thereby treating a subject with a mesothelioma. In anotherembodiment, the mesothelioma is a malignant mesothelioma. Eachpossibility represents a separate embodiment of the present invention.

In another embodiment, the present invention provides a method ofreducing an incidence of a mesothelioma, or its relapse, in a subject,the method comprising the step of administering to the subject animmunogenic composition comprising (a) a WT-1 protein; or (b) a fragmentof a WT protein, thereby reducing an incidence of a mesothelioma, or itsrelapse, in a subject. In another embodiment, the mesothelioma is amalignant mesothelioma. Each possibility represents a separateembodiment of the present invention.

In another embodiment, the present invention provides a method ofreducing an incidence of a mesothelioma, or its relapse, in a subject,the method comprising the step of administering to the subject animmunogenic composition comprising a nucleotide molecule encoding (a) aWT-1 protein; or (b) a fragment of a WT-1 protein, thereby reducing anincidence of a mesothelioma, or its relapse, in a subject. In anotherembodiment, the mesothelioma is a malignant mesothelioma. Eachpossibility represents a separate embodiment of the present invention.

In another embodiment, a target cell of an immune response elicited by amethod of the present invention presents the WT-1 peptide of the presentinvention, or a corresponding WT-1 fragment, on an HLA molecule. Inanother embodiment, the HLA molecule is an HLA class I molecule. Inother embodiments, the HLA molecule is any HLA class I subtype or HLAclass I molecule known in the art. In another embodiment, the immuneresponse against the WT-1 peptide or fragment is a heteroclitic immuneresponse. Each possibility represents a separate embodiment of thepresent invention.

In another embodiment, the WT-1-expressing cancer is an acutemyelogenous leukemia (AML). In another embodiment, the WT-1-expressingcancer is associated with a myelodysplastic syndrome (MDS). In anotherembodiment, the WT-1-expressing cancer is an MDS. In another embodiment,the WT-1-expressing cancer is a non-small cell lung cancer (NSCLC). Inanother embodiment, the WT-1-expressing cancer is a Wilms' tumor. Inanother embodiment, the WT-1-expressing cancer is a leukemia. In anotherembodiment, the WT-1-expressing cancer is a hematological cancer. Inanother embodiment, the WT-1-expressing cancer is a lymphoma. In anotherembodiment, the WT-1-expressing cancer is a desmoplastic small roundcell tumor. In another embodiment, the WT-1-expressing cancer is amesothelioma. In another embodiment, the WT-1-expressing cancer is amalignant mesothelioma. In another embodiment, the WT-1-expressingcancer is a gastric cancer. In another embodiment, the WT-1-expressingcancer is a colon cancer. In another embodiment, the WT-1-expressingcancer is a lung cancer. In another embodiment, the WT-1-expressingcancer is abreast cancer. In another embodiment, the WT-1-expressingcancer is a germ cell tumor. In another embodiment, the WT-1-expressingcancer is an ovarian cancer. In another embodiment, the WT 1-expressingcancer is a uterine cancer. In another embodiment, the WT 1-expressingcancer is a thyroid cancer. In another embodiment, the WT-1-expressingcancer is a hepatocellular carcinoma. In another embodiment, theWT-1-expressing cancer is a thyroid cancer. In another embodiment, theWT-1-expressing cancer is a liver cancer. In another embodiment, theWT-1-expressing cancer is a renal cancer. In another embodiment, theWT-1-expressing cancer is a Kaposi's sarcoma. In another embodiment, theWT-1-expressing cancer is a sarcoma. In another embodiment, theWT-1-expressing cancer is any other carcinoma or sarcoma.

In another embodiment, the WT-1-expressing cancer is a solid tumor. Inanother embodiment, the solid tumor is associated with a WT-1-expressingcancer. In another embodiment, the solid tumor is associated with amyelodysplastic syndrome (MDS). In another embodiment, the solid tumoris associated with a non-small cell lung cancer (NSCLC). In anotherembodiment, the solid tumor is associated with a lung cancer. In anotherembodiment, the solid tumor is associated with a breast cancer. Inanother embodiment, the solid tumor is associated with a colorectalcancer. In another embodiment, the solid tumor is associated with aprostate cancer. In another embodiment, the solid tumor is associatedwith an ovarian cancer. In another embodiment, the solid tumor isassociated with a renal cancer. In another embodiment, the solid tumoris associated with a pancreatic cancer. In another embodiment, the solidtumor is associated with a brain cancer. In another embodiment, thesolid tumor is associated with a gastrointestinal cancer. In anotherembodiment, the solid tumor is associated with a skin cancer. In anotherembodiment, the solid tumor is associated with a melanoma.

In another embodiment, a cancer or tumor treated by a method of thepresent invention is suspected to express WT-1. In another embodiment,WT-1 expression has not been verified by testing of the actual tumorsample. In another embodiment, the cancer or tumor is of a type known toexpress WT-1 in many cases. In another embodiment, the type expressesWT-1 in the majority of cases.

Each type of WT-1-expressing cancer or tumor, and cancer or tumorsuspected to express WT-1, represents a separate embodiment of thepresent invention.

Any embodiments enumerated herein, regarding peptides, vaccines andcompositions of this invention can be employed in any of the methods ofthis invention, and each represents an embodiment thereof.

In another embodiment, multiple peptides of this invention are used tostimulate an immune response in methods of the present invention.

The methods disclosed herein will be understood by those in the art toenable design of other WT-1-derived peptides. The methods further enabledesign of peptides binding to other HLA molecules. The methods furtherenable design of vaccines combining WT-1-derived peptides of the presentinvention. Each possibility represents a separate embodiment of thepresent invention.

In another embodiment, vaccines of the present invention have theadvantage of activating or eliciting WT-1-specific CD4⁺ T cellscontaining a variety of different HLA class II alleles. In anotherembodiment, the vaccines have the advantage of activating or elicitingWT-1-specific CD4⁺ T cells in a substantial proportion of the population(e.g. in different embodiments, 50%, 55%, 60%, 65%, 70%, 75%, 80%. 85%,90%, 95%, or greater than 95%). In another embodiment, the vaccinesactivate or elicit WT-1-specific CD4⁺ T cells in a substantialproportion of a particular population (e.g. American Caucasians). Eachpossibility represents a separate embodiment of the present invention.

In another embodiment, methods of the present invention provide for animprovement in an immune response that has already been mounted by asubject. In another embodiment, methods of the present inventioncomprise administering the peptide, composition, or vaccine 2 or moretimes. In another embodiment, the peptides are varied in theircomposition, concentration, or a combination thereof. In anotherembodiment, the peptides provide for the initiation of an immuneresponse against an antigen of interest in a subject who has not yetinitiated an immune response against the antigen. In another embodiment,the CTL that are induced proliferate in response to presentation of thepeptide on the APC or cancer cell. In other embodiments, reference tomodulation of the immune response involves, either or both the humoraland cell-mediated arms of the immune system, which is accompanied by thepresence of Th2 and Th1 T helper cells, respectively, or in anotherembodiment, each arm individually.

In other embodiments, the methods affecting the growth of a tumor resultin (1) the direct inhibition of tumor cell division, or (2) immune cellmediated tumor cell lysis, or both, which leads to a suppression in thenet expansion of tumor cells.

Inhibition of tumor growth by either of these two mechanisms can bereadily determined by one of ordinary skill in the art based upon anumber of well-known methods. In another embodiment, tumor inhibition isdetermined by measuring the actual tumor size over a period of time. Inanother embodiment, tumor inhibition can be determined by estimating thesize of a tumor (over a period of time) utilizing methods well known tothose of skill in the art. More specifically, a variety of radiologicimaging methods (e.g., single photon and positron emission computerizedtomography; see generally, “Nuclear Medicine in Clinical Oncology,”Winkler, C. (ed.) Springer-Verlag, New York, 1986), can be utilized toestimate tumor size. Such methods can also utilize a variety of imagingagents, including for example, conventional imaging agents (e.g.,Gallium-67 citrate), as well as specialized reagents for metaboliteimaging, receptor imaging, or immunologic imaging (e.g., radiolabeledmonoclonal antibody specific tumor markers). In addition,non-radioactive methods such as ultrasound (see, “UltrasonicDifferential Diagnosis of Tumors”, Kossoff and Fukuda, (eds.),Igaku-Shoin, New York, 1984), can also be utilized to estimate the sizeof a tumor.

In addition to the in vivo methods for determining tumor inhibitiondiscussed above, a variety of in vitro methods can be utilized in orderto predict in vivo tumor inhibition. Representative examples includelymphocyte mediated anti-tumor cytolytic activity determined forexample, by a ⁵¹Cr release assay (Examples), tumor dependent lymphocyteproliferation (Ioannides, et al., J. Immunol. 146(5):1700-1707, 1991),in vitro generation of tumor specific antibodies (Herlyn, et al., J.Immunol. Meth. 73:157-167, 1984), cell (e.g., CTL, helper T-cell) orhumoral (e.g., antibody) mediated inhibition of cell growth in vitro(Gazit, et al., Cancer Immunol Immunother 35:135-144, 1992), and, forany of these assays, determination of cell precursor frequency (Vose,Int. J. Cancer 30:135-142 (1982), and others.

In another embodiment, methods of suppressing tumor growth indicate agrowth state that is curtailed compared to growth without contact with,or exposure to a peptide of this invention. Tumor cell growth can beassessed by any means known in the art, including, but not limited to,measuring tumor size, determining whether tumor cells are proliferatingusing a ³H-thymidine incorporation assay, or counting tumor cells.“Suppressing” tumor cell growth refers, in other embodiments, toslowing, delaying, or stopping tumor growth, or to tumor shrinkage. Eachpossibility represents a separate embodiment of the present invention.

In another embodiment of methods and compositions of the presentinvention, WT-1 expression is measured. In another embodiment, WT-1transcript expression is measured. In another embodiment, WT-1 proteinlevels in the tumor are measured. Each possibility represents a separateembodiment of the present invention.

Methods of determining the presence and magnitude of an immune responseare well known in the art. In another embodiment, lymphocyteproliferation assays, wherein T cell uptake of a radioactive substance,e.g. ³H-thymidine is measured as a function of cell proliferation. Inother embodiments, detection of T cell proliferation is accomplished bymeasuring increases in interleukin-2 (IL-2) production, Ca²⁺ flux, ordye uptake, such as3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium. Each possibilityrepresents a separate embodiment of the present invention.

In another embodiment, CTL stimulation is determined by means known tothose skilled in the art, including, detection of cell proliferation,cytokine production and others. Analysis of the types and quantities ofcytokines secreted by T cells upon contacting ligand-pulsed targets canbe a measure of functional activity. Cytokines can be measured by ELISAor ELISPOT assays to determine the rate and total amount of cytokineproduction. (Fujihashi K. et al. (1993) J. Immunol. Meth. 160: 181;Tanguay S. and Killion J. J. (1994) Lymphokine Cytokine Res. 13:259).

In another embodiment, CTL activity is determined by ⁵¹Cr-release lysisassay. Lysis of peptide-pulsed ⁵¹Cr-labeled targets by antigen-specificT cells can be compared for target cells pulsed with control peptide. Inanother embodiment, T cells are stimulated with a peptide of thisinvention, and lysis of target cells expressing the native peptide inthe context of MHC can be determined. The kinetics of lysis as well asoverall target lysis at a fixed timepoint (e.g., 4 hours) are used, inanother embodiment, to evaluate ligand performance. (Ware C. F. et al.(1983) J Immunol 131: 1312).

Methods of determining affinity of a peptide for an HLA molecule arewell known in the art. In another embodiment, affinity is determined byTAP stabilization assays.

In another embodiment, affinity is determined by competitionradioimmunoassay. In another embodiment, the following protocol isutilized: Target cells are washed two times in PBS with 1% bovine serumalbumin (BSA; Fisher Chemicals, Fairlawn, N.J.). Cells are resuspendedat 10⁷/ml on ice, and the native cell surface bound peptides arestripped for 2 minutes at 0 [deg.] C using citrate-phosphate buffer inthe presence of 3 mg/ml beta2 microglobulin. The pellet is resuspendedat 5×10⁶ cells/ml in PBS/1% BSA in the presence of 3 mg/ml beta₂microglobulin and 30 mg/ml deoxyribonuclease, and 200 ml aliquots areincubated in the presence or absence of HLA-specific peptides for 10 minat 20° C., then with ¹²⁵I-labeled peptide for 30 min at 20° C. Totalbound ¹²⁵I is determined after two washes with PBS/2% BSA and one washwith PBS. Relative affinities are determined by comparison of escalatingconcentrations of the test peptide versus a known binding peptide.

In another embodiment, a specificity analysis of the binding of peptideto HLA on surface of live cells (e.g. SKLY-16 cells) is conducted toconfirm that the binding is to the appropriate HLA molecule and tocharacterize its restriction. This includes, in another embodiment,competition with excess unlabeled peptides known to bind to the same ordisparate HLA molecules and use of target cells which express the sameor disparate HLA types. This assay is performed, in another embodiment,on live fresh or 0.25% paraformaldehyde-fixed human PBMC, leukemia celllines and EBV-transformed T-cell lines of specific HLA types. Therelative avidity of the peptides found to bind MHC molecules on thespecific cells are assayed by competition assays as described aboveagainst ¹²⁵I-labeled peptides of known high affinity for the relevantHLA molecule, e.g., tyrosinase or HBV peptide sequence. In anotherembodiment, an HLA class II-binding peptide of methods and compositionsof the present invention is longer than the minimum length for bindingto an HLA class II molecule, which is, in another embodiment, about 12AA. In another embodiment, increasing the length of the HLA classII-binding peptide enables binding to more than one HLA class IImolecule. In another embodiment, increasing the length enables bindingto an HLA class II molecule whose binding motif is not known. In anotherembodiment, increasing the length enables binding to an HLA class Imolecule. In another embodiment, the binding motif of the HLA class Imolecule is known. In another embodiment, the binding motif of the HLAclass I molecule is not known. Each possibility represents a separateembodiment of the present invention.

In another embodiment, the peptides utilized in methods and compositionsof the present invention comprise a non-classical amino acid such as:1,2,3,4-tetrahydroisoquinoline-3-carboxylate (Kazmierski et al. (1991)J. Am Chem. Soc. 113:2275-2283); (2S,3S)-methyl-phenylalanine,(2S,3R)-methyl-phenylalanine, (2R,3S)-methyl-phenylalanine and(2R,3R)-methyl-phenylalanine (Kazmierski and Hruby (1991) TetrahedronLett. 32(41): 5769-5772); 2-aminotetrahydronaphthalene-2-carboxylic acid(Landis (1989) Ph.D. Thesis, University of Arizona);hydroxy-1,2,3,4-tetrahydroisoquinoline-3-carboxylate (Miyake et al.(1984) J. TakedaRes. Labs. 43:53-76) histidine isoquinoline carboxylicacid (Zechel et al. (1991) Int. J. Pep. Protein Res. 38(2):131-138); andHIC (histidine cyclic urea), (Dharanipragada et al (1993) Int. J. Pep.Protein Res.42(1):68-77) and ((1992) Acta. Crst., Crystal Struc. Comm48(IV): 1239-124).

In another embodiment, a peptide of this invention comprises an AAanalog or peptidomimetic, which, in other embodiments, induces or favorsspecific secondary structures. Such peptides comprise, in otherembodiments, the following: LL-Acp(LL-3-amino-2-propenidone-6-carboxylic acid), a [beta]-turn inducingdipeptide analog (Kemp et al. (1985) J. Org. Chem. 50:5834-5838);[beta]-sheet inducing analogs (Kemp et al. (1988) Tetrahedron Lett.29:5081-5082); [beta]-turn inducing analogs (Kemp et al. (1988)Tetrahedron Left. 29:5057-5060); alpha-helix inducing analogs (Kemp etal. (1988) Tetrahedron Left. 29:4935-4938); gamma-turn inducing analogs(Kemp et al. (1989) J. Org. Chem. 54:109:115); analogs provided by thefollowing references: Nagai and Sato (1985) Tetrahedron Left.26:647-650; and DiMaio et al. (1989) J. Chem. Soc. Perkin Trans, p.1687; a Gly-Ala turn analog (Kahn et al. (1989) Tetrahedron Lett.30:2317); amide bond isostere (Jones et al. (1988) Tetrahedron Left.29(31):3853-3856); tretrazol (Zabrocki et al. (1988) J. Am. Chem. Soc.110:5875-5880); DTC (Samanen et al. (1990) Int. J. Protein Pep. Res.35:501:509); and analogs taught in Olson et al. (1990) J. Am. Chem. Sci.112:323-333 and Garvey et al. (1990) J. Org. Chem. 55(3):936-940.Conformationally restricted mimetics of beta turns and beta bulges, andpeptides containing them, are described in U.S. Pat. No. 5,440,013,issued Aug. 8, 1995 to Kahn.

In other embodiments, a peptide of this invention is conjugated to oneof various other molecules, as described hereinbelow, which can be viacovalent or non-covalent linkage (complexed), the nature of whichvaries, in another embodiment, depending on the particular purpose. Inanother embodiment, the peptide is covalently or non-covalentlycomplexed to a macromolecular carrier, (e.g. an immunogenic carrier),including, but not limited to, natural and synthetic polymers, proteins,polysaccharides, polypeptides (amino acids), polyvinyl alcohol,polyvinyl pyrrolidone, and lipids. In another embodiment, a peptide ofthis invention is linked to a substrate. In another embodiment, thepeptide is conjugated to a fatty acid, for introduction into a liposome(U.S. Pat. No. 5,837,249). In another embodiment, a peptide of theinvention is complexed covalently or non-covalently with a solidsupport, a variety of which are known in the art. In another embodiment,linkage of the peptide to the carrier, substrate, fatty acid, or solidsupport serves to increase an elicited an immune response.

In other embodiments, the carrier is thyroglobulin, an albumin (e.g.human serum albumin), tetanus toxoid, polyamino acids such as poly(lysine: glutamic acid), an influenza protein, hepatitis B virus coreprotein, keyhole limpet hemocyanin, an albumin, or another carrierprotein or carrier peptide; hepatitis B virus recombinant vaccine, or anAPC. Each possibility represents a separate embodiment of the presentinvention.

In another embodiment, the term “amino acid” (AA) refers to a naturalor, in another embodiment, an unnatural or synthetic AA, and caninclude, in other embodiments, glycine, D- or L optical isomers, AAanalogs, peptidomimetics, or combinations thereof.

In another embodiment, the terms “cancer,” “neoplasm,” “neoplastic” or“tumor,” are used interchangeably and refer to cells that have undergonea malignant transformation that makes them pathological to the hostorganism. Primary cancer cells (that is, cells obtained from near thesite of malignant transformation) can be readily distinguished fromnon-cancerous cells by well-established techniques, particularlyhistological examination. The definition of a cancer cell, as usedherein, includes not only a primary cancer cell, but also any cellderived from a cancer cell ancestor. This includes metastasized cancercells, and in vitro cultures and cell lines derived from cancer cells.In another embodiment, a tumor is detectable on the basis of tumor mass;e.g., by such procedures as CAT scan, magnetic resonance imaging (MRI),X-ray, ultrasound or palpation, and in another embodiment, is identifiedby biochemical or immunologic findings, the latter which is used toidentify cancerous cells, as well, in other embodiments.

Methods for synthesizing peptides are well known in the art. In anotherembodiment, the peptides of this invention are synthesized using anappropriate solid-state synthetic procedure (see for example, Stewardand Young, Solid Phase Peptide Synthesis, Freemantle, San Francisco,Calif. (1968); Merrifield (1967) Recent Progress in Hormone Res 23:451). The activity of these peptides is tested, in other embodiments,using assays as described herein.

In another embodiment, the peptides of this invention are purified bystandard methods including chromatography (e.g., ion exchange, affinity,and sizing column chromatography), centrifugation, differentialsolubility, or by any other standard technique for protein purification.In another embodiment, immuno-affinity chromatography is used, wherebyan epitope is isolated by binding it to an affinity column comprisingantibodies that were raised against that peptide, or a related peptideof the invention, and were affixed to a stationary support.

In another embodiment, affinity tags such as hexa-His (Invitrogen),Maltose binding domain (New England Biolabs), influenza coat sequence(Kolodziej et al. (1991) Meth. Enzymol. 194:508-509),glutathione-S-transferase, or others, are attached to the peptides ofthis invention to allow easy purification by passage over an appropriateaffinity column Isolated peptides can also be physically characterized,in other embodiments, using such techniques as proteolysis, nuclearmagnetic resonance, and x-ray crystallography.

In another embodiment, the peptides of this invention are produced by invitro translation, through known techniques, as will be evident to oneskilled in the art. In another embodiment, the peptides aredifferentially modified during or after translation, e.g., byphosphorylation, glycosylation, cross-linking, acylation, proteolyticcleavage, linkage to an antibody molecule, membrane molecule or otherligand, (Ferguson et al. (1988) Ann Rev. Biochem. 57:285-320).

In another embodiment, the peptides of this invention further comprise adetectable label, which in another embodiment, is fluorescent, or inanother embodiment, luminescent, or in another embodiment, radioactive,or in another embodiment, electron dense. In other embodiments, thedetectable label comprises, for example, green fluorescent protein(GFP), DS-Red (red fluorescent protein), secreted alkaline phosphatase(SEAP), beta-galactosidase, luciferase, ³²P, ¹²⁵I, ³H and ¹⁴C,fluorescein and its derivatives, rhodamine and its derivatives, dansyland umbelliferone, luciferin or any number of other such labels known toone skilled in the art. The particular label used will depend upon thetype of immunoassay used.

In another embodiment, a peptide of this invention is linked to asubstrate, which, in another embodiment, serves as a carrier. In anotherembodiment, linkage of the peptide to a substrate serves to increase anelicited an immune response.

In another embodiment, peptides of this invention are linked to othermolecules, as described herein, using conventional cross-linking agentssuch as carbodiimide. Examples of carbodiimide are1-cyclohexyl-3-(2-morpholinyl-(4-ethyl) carbodiimide (CMC),1-ethyl-3-(3-dimethyaminopropyl) carbodiimide (EDC) and1-ethyl-3-(4-azonia-44-dimethylpentyl) carbodiimide

In other embodiments, the cross-linking agents comprise cyanogenbromide, glutaraldehyde and succinic anhydride. In general, any of anumber of homo-bifunctional agents including a homo-bifunctionalaldehyde, a homo-bifunctional epoxide, a homo-bifunctional imido-ester,a homo-bifunctional N-hydroxysuccinimide ester, a homo-bifunctionalmaleimide, a homo-bifunctional alkyl halide, a homo-bifunctional pyridyldisulfide, a homo-bifunctional aryl halide, a homo-bifunctionalhydrazide, a homo-bifunctional diazonium derivative and ahomo-bifunctional photoreactive compound can be used. Also envisioned,in other embodiments, are hetero-bifunctional compounds, for example,compounds having an amine-reactive and a sulfhydryl-reactive group,compounds with an amine-reactive and a photoreactive group and compoundswith a carbonyl-reactive and a sulfhydryl-reactive group.

In other embodiments, the homo-bifunctional cross-linking agents includethe bifunctional N-hydroxysuccinimide estersdithiobis(succinimidylpropionate), disuccinimidyl suberate, anddisuccinimidyl tartarate; the bifunctional imido-esters dimethyladipimidate, dimethyl pimelimidate, and dimethyl suberimidate; thebifunctional sulfhydryl-reactive crosslinkers1,4-di-[3′-(2′-pyridyldithio)propionamido]butane, bismaleimidohexane,and bis-N-maleimido-1,8-octane; the bifunctional aryl halides1,5-difluoro-2,4-dinitrobenzene and4,4′-difluoro-3,3′-dinitrophenylsulfone; bifunctional photoreactiveagents such as bis4b-(4-azidosalicylamido)ethyl]disulfide; thebifunctional aldehydes formaldehyde, malondialdehyde, succinaldehyde,glutaraldehyde, and adipaldehyde; a bifunctional epoxide such as1,4-butaneodiol diglycidyl ether; the bifunctional hydrazides adipicacid dihydrazide, carbohydrazide, and succinic acid dihydrazide; thebifunctional diazoniums o-tolidine, diazotized and bis-diazotizedbenzidine; the bifunctional alkylhalidesN,N′-ethylene-bis(iodoacetamide), N1N′-hexamethylene-bis(iodoacetamide),N,N′-undecamethylene-bis(iodoacetamide), as well as benzylhalides andhalomustards, such as a1a′-diiodo-p-xylene sulfonic acid andtri(2-chloroethyl)amine, respectively,

In other embodiments, hetero-bifunctional cross-linking agents used tolink the peptides to other molecules, as described herein, include, butare not limited to, SMCC(succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate), MB S(m-maleimidobenzoyl-N-hydroxysuccinimide ester), SIAB (N-succinimidyl(4-iodoacteyl)aminobenzoate), SMPB(succinimidyl-4-(p-maleimidophenyl)butyrate), GMBS(N-(.gamma.-maleimidobutyryloxy)succmimide ester), MPBH(4-(4-N-maleimidopohenyl) butyric acid hydrazide), M2C2H(4-(N-maleimidomethyl) cyclohexane-1-carboxyl-hydrazide), SMPT(succinimidyloxycarbonyl-a-methyl-a-(2-pyridyldithio)toluene), and SPDP(N-succinimidyl 3-(2-pyridyldithio)propionate).

In another embodiment, the peptides of the invention are formulated asnon-covalent attachment of monomers through ionic, adsorptive, orbiospecific interactions. Complexes of peptides with highly positivelyor negatively charged molecules can be accomplished, in anotherembodiment, through salt bridge formation under low ionic strengthenvironments, such as in deionized water. Large complexes can becreated, in another embodiment, using charged polymers such aspoly-(L-glutamic acid) or poly-(L-lysine), which contain numerousnegative and positive charges, respectively. In another embodiment,peptides are adsorbed to surfaces such as microparticle latex beads orto other hydrophobic polymers, forming non-covalently associatedpeptide-superantigen complexes effectively mimicking cross-linked orchemically polymerized protein, in other embodiments. In anotherembodiment, peptides are non-covalently linked through the use ofbiospecific interactions between other molecules. For instance,utilization of the strong affinity of biotin for proteins such as avidinor streptavidin or their derivatives could be used to form peptidecomplexes. The peptides, according to this aspect, and in anotherembodiment, can be modified to possess biotin groups using commonbiotinylation reagents such as the N-hydroxysuccinimidyl ester ofD-biotin (NHS-biotin), which reacts with available amine groups.

In another embodiment, a peptide of the present invention is linked to acarrier. In another embodiment, the carrier is KLH. In otherembodiments, the carrier is any other carrier known in the art,including, for example, thyroglobulin, albumins such as human serumalbumin, tetanus toxoid, polyamino acids such as poly (lysine:glutamicacid), influenza, hepatitis B virus core protein, hepatitis B virusrecombinant vaccine and the like. Each possibility represents a separateembodiment of the present invention.

In another embodiment, the peptides of this invention are conjugated toa lipid, such as P3 CSS. In another embodiment, the peptides of thisinvention are conjugated to a bead.

In another embodiment, the compositions of this invention furthercomprise immunomodulating compounds. In other embodiments, theimmunomodulating compound is a cytokine, chemokine, or complementcomponent that enhances expression of immune system accessory oradhesion molecules, their receptors, or combinations thereof. In someembodiments, the immunomodulating compound include interleukins, forexample interleukins 1 to 15, interferons alpha, beta or gamma, tumournecrosis factor, granulocyte-macrophage colony stimulating factor(GM-CSF), macrophage colony stimulating factor (M-CSF), granulocytecolony stimulating factor (G-CSF), chemokines such as neutrophilactivating protein (NAP), macrophage chemoattractant and activatingfactor (MCAF), RANTES, macrophage inflammatory peptides MIP-Ia andMIP-Ib, complement components, or combinations thereof. In otherembodiments, the immunomodulating compound stimulate expression, orenhanced expression of OX40, OX40L (gp34), lymphotactin, CD40, CD40L,B7.1, B7.2, TRAP, ICAM-1, 2 or 3, cytokine receptors, or combinationthereof.

In another embodiment, the immunomodulatory compound induces or enhancesexpression of co-stimulatory molecules that participate in the immuneresponse, which include, in some embodiments, CD40 or its ligand, CD28,CTLA-4 or a B7 molecule. In another embodiment, the immunomodulatorycompound induces or enhances expression of a heat stable antigen (HSA)(Liu Y. et al. (1992) J. Exp. Med. 175:437-445), chondroitinsulfate-modified MHC invariant chain (Ii-CS) (Naujokas M. F. et al(1993) Cell 74:257-268), or an intracellular adhesion molecule 1(ICAM-I) (Van R. H. (1992) Cell 71: 1065-1068), which assists, inanother embodiment, co-stimulation by interacting with their cognateligands on the T cells.

In another embodiment, the composition comprises a solvent, includingwater, dispersion media, cell culture media, isotonic agents and thelike. In another embodiment, the solvent is an aqueous isotonic bufferedsolution with a pH of around 7.0. In another embodiment, the compositioncomprises a diluent such as water, phosphate buffered saline, or saline.In another embodiment, the composition comprises a solvent, which isnon-aqueous, such as propyl ethylene glycol, polyethylene glycol andvegetable oils.

In another embodiment, the composition is formulated for administrationby any of the many techniques known to those of skill in the art. Forexample, this invention provides for administration of thepharmaceutical composition parenterally, intravenously, subcutaneously,intradermally, intramucosally, topically, orally, or by inhalation.

In another embodiment, the vaccine comprising a peptide of thisinvention further comprises a cell population, which, in anotherembodiment, comprises lymphocytes, monocytes, macrophages, dendriticcells, endothelial cells, stem cells or combinations thereof, which, inanother embodiment are autologous, syngeneic or allogeneic, with respectto each other. In another embodiment, the cell population comprises apeptide of the present invention. In another embodiment, the cellpopulation takes up the peptide. Each possibility represents a separateembodiment of the present invention.

In another embodiment, the cell populations of this invention areobtained from in vivo sources, such as, for example, peripheral blood,leukopheresis blood product, apheresis blood product, peripheral lymphnodes, gut associated lymphoid tissue, spleen, thymus, cord blood,mesenteric lymph nodes, liver, sites of immunologic lesions, e.g.synovial fluid, pancreas, cerebrospinal fluid, tumor samples,granulomatous tissue, or any other source where such cells can beobtained. In another embodiment, the cell populations are obtained fromhuman sources, which are, in other embodiments, from human fetal,neonatal, child, or adult sources. In another embodiment, the cellpopulations of this invention are obtained from animal sources, such as,for example, porcine or simian, or any other animal of interest. Inanother embodiment, the cell populations of this invention are obtainedfrom subjects that are normal, or in another embodiment, diseased, or inanother embodiment, susceptible to a disease of interest.

In another embodiment, the cell populations of this invention areseparated via affinity-based separation methods. Techniques for affinityseparation include, in other embodiments, magnetic separation, usingantibody-coated magnetic beads, affinity chromatography, cytotoxicagents joined to a monoclonal antibody or use in conjunction with amonoclonal antibody, for example, complement and cytotoxins, and“panning” with an antibody attached to a solid matrix, such as a plate,or any other convenient technique. In other embodiment, separationtechniques include the use of fluorescence activated cell sorters, whichcan have varying degrees of sophistication, such as multiple colorchannels, low angle and obtuse light scattering detecting channels,impedance channels, etc. In other embodiments, any technique thatenables separation of the cell populations of this invention can beemployed, and is to be considered as part of this invention.

In another embodiment, the dendritic cells are from the diversepopulation of morphologically similar cell types found in a variety oflymphoid and non-lymphoid tissues, qualified as such (Steinman (1991)Ann Rev. Immunol. 9:271-296). In another embodiment, the dendritic cellsused in this invention are isolated from bone marrow, or in anotherembodiment, derived from bone marrow progenitor cells, or, in anotherembodiment, from isolated from/derived from peripheral blood, or inanother embodiment, derived from, or are a cell line.

In another embodiment, the cell populations described herein areisolated from the white blood cell fraction of a mammal, such as amurine, simian or a human (See, e.g., WO 96/23060). The white blood cellfraction can be, in another embodiment, isolated from the peripheralblood of the mammal

Methods of isolating dendritic cells are well known in the art. Inanother embodiment, the DC are isolated via a method which includes thefollowing steps: (a) providing a white blood cell fraction obtained froma mammalian source by methods known in the art such as leukophoresis;(b) separating the white blood cell fraction of step (a) into four ormore subfractions by countercurrent centrifugal elutriation; (c)stimulating conversion of monocytes in one or more fractions from step(b) to dendritic cells by contacting the cells with calcium ionophore,GM-CSF and IL-13 or GM-CSF and IL-4, (d) identifying the dendriticcell-enriched fraction from step (c); and (e) collecting the enrichedfraction of step (d), preferably at about 4[deg.] C.

In another embodiment, the dendritic cell-enriched fraction isidentified by fluorescence-activated cell sorting, which identifies atleast one of the following markers: HLA-DR, HLA-DQ, or B7.2, and thesimultaneous absence of the following markers: CD3, CD14, CD16, 56, 57,and CD 19, 20.

In another embodiment, the cell population comprises lymphocytes, whichare, in another embodiment, T cells, or in another embodiment, B cells.The T cells are, in other embodiments, characterized as NK cells, helperT cells, cytotoxic T lymphocytes (CTL), TBLs, native T cells, orcombinations thereof. It is to be understood that T cells which areprimary, or cell lines, clones, etc. are to be considered as part ofthis invention. In another embodiment, the T cells are CTL, or CTLlines, CTL clones, or CTLs isolated from tumor, inflammatory, or otherinfiltrates.

In another embodiment, hematopoietic stem or early progenitor cellscomprise the cell populations used in this invention. In anotherembodiment, such populations are isolated or derived, by leukaphoresis.In another embodiment, the leukapheresis follows cytokineadministration, from bone marrow, peripheral blood (PB) or neonatalumbilical cord blood. In another embodiment, the stem or progenitorcells are characterized by their surface expression of the surfaceantigen marker known as CD34^(±), and exclusion of expression of thesurface lineage antigen markers, Lin-.

In another embodiment, the subject is administered a peptide,composition or vaccine of this invention, in conjunction with bonemarrow cells. In another embodiment, the administration together withbone marrow cells embodiment follows previous irradiation of thesubject, as part of the course of therapy, in order to suppress, inhibitor treat cancer in the subject.

In another embodiment, the phrase “contacting a cell” or “contacting apopulation” refers to a method of exposure, which can be, in otherembodiments, direct or indirect. In another embodiment, such contactcomprises direct injection of the cell through any means well known inthe art, such as microinjection. It is also envisaged, in anotherembodiment, that supply to the cell is indirect, such as via provisionin a culture medium that surrounds the cell, or administration to asubject, via any route well known in the art, and as described herein.

In another embodiment, CTL generation of methods of the presentinvention is accomplished in vivo, and is effected by introducing into asubject an antigen presenting cell contacted in vitro with a peptide ofthis invention (See for example Paglia et al. (1996) J. Exp. Med.183:317-322).

In another embodiment, the peptides of methods and compositions of thepresent invention are delivered to APC. In another embodiment, thepeptide-pulsed APC are administered to a subject to elicit and immuneresponse or treat or inhibit growth or recurrence of a tumor. Eachpossibility represents a separate embodiment of the present invention.

In another embodiment, the peptides are delivered to APC in the form ofcDNA encoding the peptides. In another embodiment, the term“antigen-presenting cells” (APC) refers to dendritic cells (DC),monocytes/macrophages, B lymphocytes or other cell type(s) expressingthe necessary MHC/co-stimulatory molecules, which effectively allow forT cell recognition of the presented peptide. In another embodiment, theAPC is a cancer cell. Each possibility represents a separate embodimentof the present invention.

In another embodiment, the CTL are contacted with 2 or more APCpopulations. In another embodiment, the 2 or more APC populationspresent different peptides. Each possibility represents a separateembodiment of the present invention.

In another embodiment, techniques that lead to the expression of antigenin the cytosol of APC (e.g. DC) are used to deliver the peptides to theAPC. Methods for expressing antigens on APC are well known in the art.In another embodiment, the techniques include (1) the introduction intothe APC of naked DNA encoding a peptide of this invention, (2) infectionof APC with recombinant vectors expressing a peptide of this invention,and (3) introduction of a peptide of this invention into the cytosol ofan APC using liposomes. (See Boczkowski D. et al. (1996) J. Exp. Med.184:465-472; Rouse et al. (1994) J. Virol. 68:5685-5689; and Nair et al.(1992) J. Exp. Med. 175:609-612).

In another embodiment, foster APC such as those derived from the humancell line 174xCEM.T2, referred to as T2, which contains a mutation inits antigen processing pathway that restricts the association ofendogenous peptides with cell surface MHC class I molecules (Zweerink etal. (1993) J. Immunol. 150:1763-1771), are used, as exemplified herein.

In another embodiment, as described herein, the subject is exposed to apeptide, or a composition/cell population comprising a peptide of thisinvention, which differs from the native protein expressed, whereinsubsequently a host immune cross-reactive with the nativeprotein/antigen develops.

In another embodiment, the subject, as referred to in any of the methodsor embodiments of this invention is a human. In other embodiments, thesubject is a mammal, which can be a mouse, rat, rabbit, hamster, guineapig, horse, cow, sheep, goat, pig, cat, dog, monkey, or ape. Eachpossibility represents a separate embodiment of the present invention.

In another embodiment, peptides, vaccines, and compositions of thisinvention stimulate an immune response that results in tumor cell lysis.

In another embodiment, any of the methods described herein is used toelicit CTL, which are elicited in vitro. In another embodiment, the CTLare elicited ex-vivo. In another embodiment, the CTL are elicited invitro. The resulting CTL, are, in another embodiment, administered tothe subject, thereby treating the condition associated with the peptide,an expression product comprising the peptide, or a homologue thereof.Each possibility represents a separate embodiment of the presentinvention.

In another embodiment, the method entails introduction of the geneticsequence that encodes the peptides of this invention using, e.g., one ormore nucleic acid delivery techniques. Nucleic acids of the inventioninclude, in another embodiment, DNA, RNA and mixtures of DNA and RNA,alone or in conjunction with non-nucleic acid components. In anotherembodiment, the method comprises administering to the subject a vectorcomprising a nucleotide sequence, which encodes a peptide of the presentinvention (Tindle, R. W. et al. Virology (1994) 200:54). In anotherembodiment, the method comprises administering to the subject naked DNAwhich encodes a peptide, or in another embodiment, two or more peptidesof this invention (Nabel, et al. PNAS-USA (1990) 90: 11307). In anotherembodiment, multi-epitope, analogue-based cancer vaccines are utilized(Pikes et al, Design of multi-epitope, analogue-based cancer vaccines.Expert Opin Biol Ther. 2003 September; 3(6):985-93). Each possibilityrepresents a separate embodiment of the present invention.

Nucleic acids can be administered to a subject via any means as is knownin the art, including parenteral or intravenous administration, or inanother embodiment, by means of a gene gun. In another embodiment, thenucleic acids are administered in a composition, which correspond, inother embodiments, to any embodiment listed herein.

Vectors for use according to methods of this invention can comprise anyvector that facilitates or allows for the expression of a peptide ofthis invention. Vectors comprise, in some embodiments, attenuatedviruses, such as vaccinia or fowlpox, such as described in, e.g., U.S.Pat. No. 4,722,848, incorporated herein by reference. In anotherembodiment, the vector is BCG (Bacille Calmette Guerin), such asdescribed in Stover et al. (Nature 351:456-460 (1991)). A wide varietyof other vectors useful for therapeutic administration or immunizationof the peptides of the invention, e.g., Salmonella typhi vectors and thelike, will be apparent to those skilled in the art from the descriptionherein.

In another embodiment, the vector further encodes for animmunomodulatory compound, as described herein. In another embodiment,the subject is administered an additional vector encoding same,concurrent, prior to or following administration of the vector encodinga peptide of this invention to the subject.

In another embodiment, the peptides, compositions and vaccines of thisinvention are administered to a subject, or utilized in the methods ofthis invention, in combination with other anticancer compounds andchemotherapeutics, including monoclonal antibodies directed againstalternate cancer antigens, or, in another embodiment, epitopes thatconsist of an AA sequence which corresponds to, or in part to, that fromwhich the peptides of this invention are derived.

Various embodiments of dosage ranges are contemplated by this invention.In another embodiment, the dosage is 20 μg per peptide per day. Inanother embodiment, the dosage is 10 μg/peptide/day. In anotherembodiment, the dosage is 30 μg/peptide/day. In another embodiment, thedosage is 40 μg/peptide/day. In another embodiment, the dosage is 60μg/peptide/day. In another embodiment, the dosage is 80 μg/peptide/day.In another embodiment, the dosage is 100 μg/peptide/day. In anotherembodiment, the dosage is 150 μg/peptide/day. In another embodiment, thedosage is 200 μg/peptide/day. In another embodiment, the dosage is 300μg/peptide/day. In another embodiment, the dosage is 400 μg/peptide/day.In another embodiment, the dosage is 600 μg/peptide/day. In anotherembodiment, the dosage is 800 μg/peptide/day. In another embodiment, thedosage is 1000 μg/peptide/day. In another embodiment, the dosage is 1500μg/peptide/day. In another embodiment, the dosage is 2000μg/peptide/day.

In another embodiment, the dosage is 10 μg/peptide/dose. In anotherembodiment, the dosage is 30 μg/peptide/dose. In another embodiment, thedosage is 40 μg/peptide/dose. In another embodiment, the dosage is 60μg/peptide/dose. In another embodiment, the dosage is 80μg/peptide/dose. In another embodiment, the dosage is 100μg/peptide/dose. In another embodiment, the dosage is 150μg/peptide/dose. In another embodiment, the dosage is 200μg/peptide/dose. In another embodiment, the dosage is 300μg/peptide/dose. In another embodiment, the dosage is 400μg/peptide/dose. In another embodiment, the dosage is 600μg/peptide/dose. In another embodiment, the dosage is 800μg/peptide/dose. In another embodiment, the dosage is 1000μg/peptide/dose. In another embodiment, the dosage is 1500μg/peptide/dose. In another embodiment, the dosage is 2000μg/peptide/dose.

In another embodiment, the dosage is 10-20 μg/peptide/dose. In anotherembodiment, the dosage is 20-30 μg/peptide/dose. In another embodiment,the dosage is 20-40 μg/peptide/dose. In another embodiment, the dosageis 30-60 μg/peptide/dose. In another embodiment, the dosage is 40-80μg/peptide/dose. In another embodiment, the dosage is 50-100μg/peptide/dose. In another embodiment, the dosage is 50-150μg/peptide/dose. In another embodiment, the dosage is 100-200μg/peptide/dose. In another embodiment, the dosage is 200-300μg/peptide/dose. In another embodiment, the dosage is 300-400μg/peptide/dose. In another embodiment, the dosage is 400-600μg/peptide/dose. In another embodiment, the dosage is 500-800μg/peptide/dose. In another embodiment, the dosage is 800-1000μg/peptide/dose. In another embodiment, the dosage is 1000-1500μg/peptide/dose. In another embodiment, the dosage is 1500-2000μg/peptide/dose.

In another embodiment, the total amount of peptide per dose or per dayis one of the above amounts. In another embodiment, the total peptidedose per dose is one of the above amounts.

Each of the above doses represents a separate embodiment of the presentinvention.

Various embodiments of dosage ranges are contemplated by this invention.In another embodiment, the dosage is 20 mg per peptide per day. Inanother embodiment, the dosage is 10 mg/peptide/day. In anotherembodiment, the dosage is 30 mg/peptide/day. In another embodiment, thedosage is 40 mg/peptide/day. In another embodiment, the dosage is 60mg/peptide/day. In another embodiment, the dosage is 80 mg/peptide/day.In another embodiment, the dosage is 100 mg/peptide/day. In anotherembodiment, the dosage is 150 mg/peptide/day. In another embodiment, thedosage is 200 mg/peptide/day. In another embodiment, the dosage is 300mg/peptide/day. In another embodiment, the dosage is 400 mg/peptide/day.In another embodiment, the dosage is 600 mg/peptide/day. In anotherembodiment, the dosage is 800 mg/peptide/day. In another embodiment, thedosage is 1000 mg/peptide/day.

In another embodiment, the dosage is 10 mg/peptide/dose. In anotherembodiment, the dosage is 30 mg/peptide/dose. In another embodiment, thedosage is 40 mg/peptide/dose. In another embodiment, the dosage is 60mg/peptide/dose. In another embodiment, the dosage is 80mg/peptide/dose. In another embodiment, the dosage is 100mg/peptide/dose. In another embodiment, the dosage is 150mg/peptide/dose. In another embodiment, the dosage is 200mg/peptide/dose. In another embodiment, the dosage is 300mg/peptide/dose. In another embodiment, the dosage is 400mg/peptide/dose. In another embodiment, the dosage is 600mg/peptide/dose. In another embodiment, the dosage is 800mg/peptide/dose. In another embodiment, the dosage is 1000mg/peptide/dose.

In another embodiment, the dosage is 10-20 mg/peptide/dose. In anotherembodiment, the dosage is 20-30 mg/peptide/dose. In another embodiment,the dosage is 20-40 mg/peptide/dose. In another embodiment, the dosageis 30-60 mg/peptide/dose. In another embodiment, the dosage is 40-80mg/peptide/dose. In another embodiment, the dosage is 50-100mg/peptide/dose. In another embodiment, the dosage is 50-150mg/peptide/dose. In another embodiment, the dosage is 100-200mg/peptide/dose. In another embodiment, the dosage is 200-300mg/peptide/dose. In another embodiment, the dosage is 300-400mg/peptide/dose. In another embodiment, the dosage is 400-600mg/peptide/dose. In another embodiment, the dosage is 500-800mg/peptide/dose. In another embodiment, the dosage is 800-1000mg/peptide/dose.

In another embodiment, the total amount of peptide per dose or per dayis one of the above amounts. In another embodiment, the total peptidedose per dose is one of the above amounts.

Each of the above doses represents a separate embodiment of the presentinvention.

In another embodiment, the present invention provides a kit comprising apeptide, composition or vaccine of the present invention. In anotherembodiment, the kit further comprises a label or packaging insert. Inanother embodiment, the kit is used for detecting a WT-1-specific CD4response through the use of a delayed-type hypersensitivity test. Inanother embodiment, the kit is used for any other method enumeratedherein. In another embodiment, the kit is used for any other methodknown in the art. Each possibility represents a separate embodiment ofthe present invention.

Among those antigens uniquely or differentially expressed by malignantcells, WT-1 is considered one of the most promising (47). However, thenumber of immunogenic WT-1 peptide antigens previously identified andreported is very limited, and largely confined to a set of peptidespresented by the HLA alleles A0201, A2402 and DRB 10401. As will be seenfrom the examples presented below, using a pool of overlapping 15-merpeptides spanning the amino acid sequence of WT-1 loaded on autologousAPCs for sensitization, WT-1 peptide-specific IFNγ+CD4+ and CD8 T-cellresponses were generated from the blood of 41/56 (78%) normal donors,and thereafter the epitopes eliciting these responses and theirpresenting HLA alleles were identified. Of the 42 WT-1 peptide antigensdescribed, all but one have not been heretofore identified. The newimmunogenic peptides identified include 36 peptides presented by class IHLA alleles and 5 presented by class II HLA alleles. Of the peptidespresented by class I HLA alleles, 10 nonamer epitopes were identifiedwhich could be presented by from 2-4 different HLA alleles. Alsoidentified, within 4 pentadecapeptides, were overlapping 11-mer andnonamer sequences that co-induced distinguishable CD4+IFNγ+ andCD8+IFNγ+ T-cells. Whether and to what degree epitopes that can bepresented by more than one allele can elicit enhanced WT-1 specificresponses in individuals inheriting both presenting HLA alleles or boththe class I and class II presenting HLA alleles in those instances inwhich overlapping sequences are contained in the same 15-mer is readilydeterminable; however, inclusion of such peptides in WT-1 vaccines couldsignificantly broaden their applicability particularly among patientsnot inheriting HLA-A0201 or A2402.

As shown in the examples, those peptides presented by class I HLAalleles elicited IFNγ+CD8+ T-cells that were able to lyse peptide loadedautologous APCs as well as allogeneic APCs sharing the T-cells'restricting HLA allele in 50/51 (99%) and 48/51 (94%) cultures testedrespectively (Table 1, 2). More importantly, of 36 HLA-restricted WT-1peptide specific T-cell lines that could be tested, T-cell linesspecific for 29 epitopes including 2/4 epitopes presented by class IIand 27/32 presented by class I alleles, were also able to lyse WT-1+leukemic blasts sharing the T-cells' restricting HLA allele. The failureof the HLA-restricted WT-1 epitope-specific T-cells to lyse allogeneicPHA blasts from the same leukemic patients (Table 3A), coupled with thedifferential leukemocidal activity of T-cells sensitized with WT-1peptide-loaded autologous EBVBLCL when compared to aliquots of the sameT-cells sensitized with autologous EBVBLCL alone (Table 3B) indicatesthat the leukemocidal activity is WT-1 peptide-specific and not a resultof contaminating alloreactive T-cells. Thus, these data show that 29/36immunogenic peptides of WT-1 identified (80%) can be processed andpresented by WT-1+ leukemic cells at concentrations adequate for WT-1epitope-specific T-cell recognition and cytolysis.

In FIG. 4, maps are shown of the WT-1 protein. FIG. 4C defines thelocalization of all previously reported antigenic epitopes presented byHLA class I and II alleles; FIG. 4D depicts the location of immunogenicpeptides identified in this report. As can be seen, the 11 epitopespreviously reported to be presented by class I and 10 presented by classII HLA alleles are principally clustered in sequences encoded by exons1, 7 and 10, while the epitopes recognized by normal T-cells sensitizedwith the WT-1 peptide pool are principally clustered in sequencesencoded by the first 5 exons. Thus, 26 of the new epitopes are includedin each of the four major isoforms of WT-1 resulting from splicevariants that do or do not include the 17 amino acid sequence (aas250-266) in exon 5 or the three amino acid sequence (₄₀₀₋₄₁₀KTS) betweenzinc fingers 3 and 4. While the epitopes are broadly distributed,clusters of epitopes were detected in the RNA recognition domain in exon1 and the activation domain (aa 181-250) (FIG. 4F) proximal to thespliced 17aa segment in exon 5. The latter area also contained thoseepitopes most frequently recognized by multiple donors (FIG. 4E).Interestingly, 9 newly identified epitopes map to a 126 amino acidsequence at the N terminus encoded by a segment of the WT-1 geneinitially described by Gessler et al (37) that is centromeric to exon 1of the (Exon 5+, KTS+) isoform of WT-1 and includes the long isoform ofWT-1 initiated at a CUG codon upstream of the AUG initiator for exon1.50 Strikingly, each of the epitopes identified in this sequenceelicits IFNγ+ T-cells that are cytolytic against leukemic blastscoexpressing WT-1 and the T-cells' restricting HLA allele.

Of the several “self” proteins such as WT-1, NY-ESO-1, HER2/neu, MAGE,and others, differentially expressed by specific tumors, only WT-1 andMART-1 have been shown to elicit responses in normal donors (31, 32,51-54). In contrast, T-cells specific for each of these proteins havebeen recorded in a proportion of patients with tumors overexpressingthem (55). In particular, T-cells specific for the RMF and CMT peptidesof WT-1 have been detected in patients with leukemias, myeloma,carcinoma of the breast and prostate and other solid tumors (31, 32,56-61). Responses to several of the WT-1 epitopes identified in thepresent study in 50-60% of patients with ovarian cancer have beendocumented. Given the high number of potentially immunogenic epitopes inproteins such as NY-ESO-1 and HER2/neu that have elicited responses intumor-bearing hosts (62), the number of immunogenic WT-1 peptides wehave identified is not sufficiently different to account for thedifferential presence of WT-1 responses in normal donors. Furthermore,Pospori et al (63) have shown that HSCs expressing a transduced TCRspecific for a WT-1 peptide presented by HLA-A0201 are not deleted inthe thymus of HLA-A0201 transgenic mice and generate functional memoryT-cells. However, while the basis for this lack of “self” tolerance isunclear, the studies of Rezvani et al (31) and data herein (FIG. 1A)indicate that the frequencies of WT-1 specific T-cells in the blood ofhealthy donors is low. In part, this may reflect the low levels andlimited tissue distribution of WT-1 expression in normal individuals(18-20). Recently, Rezvani et al (64) also demonstrated declining T-cellresponses to WT-1 in patients repeatedly vaccinated with WT-1 peptides,suggesting that these responses are highly regulated. Lehe et al (65)have also recently shown that sensitization of T-cells with a WT-1peptide presented by DRB 10402 in the presence of high concentrations ofIL-2 preferentially stimulates the generation of CD25+FOXP3+GITR+CD127-regulatory T-cells capable of inhibiting CD8+WT-1 specificT-cell responses.

Under the culture conditions employed herein, autologous DCs and EBVBLCLloaded with the WT-1 peptide pool preferentially induced the generationof CD8+ and CD4+IFNγ+WT-1 peptide-specific T-cells from 41/56 normaldonors (73%). Although each donor recognized only 1-3 epitopes of WT-1,the fact that T-cells specific for 80% of these epitopes could recognizeWT-1+ leukemic cells sharing the T-cells' presenting HLA allele suggeststhat the turnover and processing of the aberrantly expressed WT-1 ishigh, permitting the simultaneous presentation of several different WT-1epitopes by the restricting HLA allele expressed by these leukemiccells. Identification of these epitopes is useful both for in vitrogeneration of potent tumoricidal WT-1 specific T-cells for adoptive celltherapies and for the generation of more broadly applicable vaccines forstimulating T-cell responses for eradication of clonogenic tumor cellsexpressing WT-1 in vivo.

In one embodiment, peptides from the WT-1 protein sequence that areupstream from exon 1, i.e., within the first 126 amino acids of SEQ IDNO:194, are heretofore unrecognized sites of immunogenic epitopes andtherefore peptides useful for the purposes herein.

Example 1 Binding of HLA-A0201 and -A0301 by Synthetic Peptide AnaloguesDerived from WT-1

Materials and Experimental Methods.

Peptides were synthesized by Genemed Synthesis Inc, CA usingfluorenylmethoxycarbonyl chemistry and solid phase synthesis, and werepurified by high pressure liquid chromatography (HPLC). The quality ofthe peptides was assessed by HPLC analysis, and the expected molecularweight was measured using matrix-assisted laser desorption massspectrometry. Peptides were sterile and >90% pure. The peptides weredissolved in DMSO and diluted in PBS at pH 7.4 or saline solution toyield a concentration of 5 milligrams per milliliter (mg/ml) and werestored at −80° C. For in vitro experiments, an irrelevant controlpeptide, HLA A24 consensus, was used.

Peptide Sequence Analysis.

Peptide sequence analysis was performed using 2 databases. The first wasthe software of the Bioinformatics & Molecular Analysis Section(National Institutes of Health, Washington, DC) (Parker K C et al,Scheme for ranking potential HLA-A2 binding peptides based onindependent binding of individual peptide side-chains. J Immunol 152:163-175, 1994), which ranks 9-mer or 10-mer peptides on a predictedhalf-time dissociation coefficient from HLA class I molecules. Thesecond database, SYFPEITHI prediction software, is described inRammensee H G et al (SYFPEITHI: database for MHC ligands and peptidemotifs. Immunogenetics 50: 213-219, 1999). Irrelevant control peptidesused in in vitro experiments were: RAS (TEYKLVVVGAPGVGKSALTIQ; SEQ IDNo: 49) or CML b2a2 (VHSIPLTINKEEALQRPVASDFE; SEQ ID No: 50) for ClassII, and HIV pol (ILKEPVHGV; SEQ ID No: 51) or CML F (YLKALQRPY; SEQ IDNo: 52) for Class I.

Cell Lines.

Cell lines were cultured in RPMI 1640 medium supplemented with 5% FCS,penicillin, streptomycin, 2 mM glutamine and 2-mercaptoethanol at 37° C.in humidified air containing 5% CO2. T2 is a human cell line lackingTAP1 and TAP2 and therefore unable to present peptides derived fromcytosolic proteins. Raji cells are a human Burkitt lymphoma cells thatexhibit a high level of TAP expression.

Human mesothelioma cell lines studied included: sarcomatoid (VAMT,H2373, H28), epithelioid (H2452) and biphasic (JMN, MSTO and H-Meso1A).Cell lines were obtained from the following sources: H-Meso1A: NCI,Bethesda, Md.; JMN and VAMT: Dr. Sirotnak, Memorial Sloan KetteringCancer Center (MSKCC); H-2452 and H2373: Dr. Pass, Karmanos CancerInstitute, Wayne State University, Detroit, Mich.; H28 and MSTO:American Type Culture Collection (ATCC, Manassas, Va.). Cell lines weremaintained in media recommended by the suppliers and incubated in ahumidified incubator with 5% CO2.

Mesothelioma cell lines Meso 11, Meso 34, Meso 37, Meso 47 and Meso 56were obtained from Dr. M Gregoire (Institute of Biology, Nantes, France)and cultured in RPMI 1640 (Life Technologies)+10% fetal calf serum(FCS), 1% penicillin-streptomycin, and 1% L-glutamine. All cells wereHLA typed by the Department of Cellular Immunology at MSKCC. Melanomacell line Mewo (WT-1-A201+) was obtained from the ATCC. SKRC-52 renalcell carcinoma was obtained from L. Old of the Ludwig Institute.Leukemia cell lines were cultured in RPMI 1640+10% FCS, 1%penicillin-streptomycin, 2 mM glutamine and 2-mercaptoethanol at 37°C./5% CO2. LAMA81, BV173 and 697, Ph+ leukemias that are all WT-1+ andA0201+, were provided by Dr. HJ Stauss (University College London).SKLY-16 is a human B cell lymphoma (WT-1−, A0201+); K562, RwLeu4 andHL60, all WT-1+ leukemias, were obtained from the ATCC.

T2 assay for peptide binding and stabilization of HLA A0201 molecules.T2 cells (TAP−, HLA-A0201⁺) were incubated overnight at 27° C. at aconcentration of 1×10⁶ cells/ml in FCS-free RPMI medium supplementedwith 5 μg/ml human B_(2m) (Sigma, St Louis, Mo.) in the absence(negative control) or presence of either a positive reference tyrosinasepeptide or test peptides at various final concentrations (50, 10, 1, and0.1 micrograms g)/m1). Following a 4-hour incubation with 5 μg/mlbrefeldin A (Sigma), T2 cells were labeled for 30 minutes at 4° C. witha saturating concentration of anti-HLA-A2.1 (BB7.2) mAb, then washedtwice. Cells were then incubated for 30 minutes, 4° C. with a saturatingconcentration of FITC-conjugated goat IgG F(ab′)2 anti-mouse Ig (Caltag,San Francisco, Calif.), washed twice, fixed in PBS/1% paraformaldehydeand analyzed using a FACS Calibur® cytofluorometer (Becton Dickinson,Immunocytometry Systems, San Jose, Calif.).

The mean intensity of fluorescence (MIF) observed for each peptideconcentration (after dividing by the MIF in the absence of peptide) wasused as an indication of peptide binding and expressed as a“fluorescence index.” Stabilization assays were performed similarly.Following initial evaluation of peptide binding at time 0, cells werewashed in RPMI complete medium to remove free peptides and incubated inthe continuous presence of 0.5 μg/ml brefeldin-A for 2, 4, 6 or 8 hours.

The number of stable peptide-HLA-A2.1 complexes was estimated asdescribed above by immunofluorescence. The half time of complexes is anestimate of the time required for a 50% reduction of the MIF value attime=0.

WT-1 Peptides.

The sequence of the WT-1 protein published by Gessler et al. (37) whichcomprises 575 aminoacids and includes the first 126 aminoacids in theN-terminus missing in the (Exon 5+, KTS+) isoform of WT-116, was used todesign the peptide sequences (SEQ ID NO:1; FIG. 2A). 141 pentadecapeptides spanning this sequence, each overlapping the next by 11aa, weresynthesized by Invitrogen (Baltimore, Md.) to specifications ofvalidated sequence, 95% purity, sterility and absence of endotoxin.These 141 15-mers were mixed in equal amounts to form a total pool ofpeptides, in which each peptide is at a concentration of 0.35mcg/ml.This pool was used for the T-cell sensitization. To identify peptideseliciting responses, subpools containing 12 pentadeca peptides(4.17mcg/ml/peptide) were established to form a mapping matrix in whicheach peptide is included in only two overlapping subpools (FIG. 2B).

Generation of WT-1 Specific T-Cells:

Peripheral blood was obtained from 56 consenting normal donors accordingto protocols approved by the Institutional Review Board of MemorialSloan-Kettering Cancer Center (New York, N.Y.). All donors were typedfor HLA-A, B, C, DR and DQ at high resolution by standard techniques.

Cytokine-activated monocytes (CAMs) were used as antigen presentingcells (APCs), and generated as previously described (32). Briefly,peripheral blood monocytes were separated by adherence on plastic andcultured in RPMI1640 containing 1% autologous serum. GM-CSF (Berlex,Montville, N.J.) and interleukin-4 (IL-4) (R&D Systems, Minneapolis,Minn.) were added to final concentrations of 2000 U/ml and 1000 U/mlrespectively on days 0, 2, 4. On day 5, these cells were additionallytreated with TNFα (10 ng/ml), interleukin-6 (IL-6) (1000 IU/ml), IL1β(400 IU/ml), PGE2 (25 mM-3) (R&D Systems, Minneapolis, Minn.) togetherwith GM-CSF and IL-4 at the same doses. CAMs harvested on day 7 ofculture expressed CD83, CD80, CD86, and HLA class I and II alleles asdetermined by FACS analysis.

EBV-BLCL were also used as WT-1 peptide loaded and control APCs or astargets as specified in the experiments. They were generated byinfection of peripheral blood mononuclear cells (PBMC) with EBV strainB95.8 (38,39) as previously described. The EBV transformed BLCL(EBV-BLCL) were cultured in RPMI1640 (Gemini) with 10% fetal calf serum(Gemini) in the presence of Acyclovir.

Sensitization and Propagation of WT-1 Specific T-Cells.

To generate WT-1-specific CTLs, PBMC were isolated by Ficoll-Hypaquedensity gradient centrifugation. Monocytes were depleted by adherence onplastic and NK cells by absorption to immunomagnetic CD56 pre-coatedmicrobeads (Miltenyi Biotech Inc, MA) as previously described (32).Enriched T-cell fractions were stimulated at a 20:1 responder:stimulatorratio with autologous CAMs or EBV-BLCL that had been pre-loaded for 3hours with the total pool of the WT-1 pentadecapeptides in serum-freemedium and irradiated to 3000 cGy. T-cells were cultured in Yssel'smedium supplemented with 5% AB human serum (YH5, Gemini), re-stimulatedweekly with the autologous WT-1 total pool-loaded CAMs or EBV-BLCL andfed with interleukin-2(IL-2) (Collaborative Biomedical Products,Bedford, Mass.) every 2-3 days at 10-50 U/ml.

Cell Targets—Leukemic Cells:

Twenty-four primary leukemic cells and 1 leukemic cell line werecharacterized for their expression of WT-1 by intracellular FACSstaining using murine anti-human WT-1 monoclonal antibodies (Neomarkers,Fremont, Calif.) as previously described (32,38) The WT-1+ leukemiasincluded blast cells from 11 primary AMLs, 3 primary ALLs and 1 B-cellprecursor ALL cell line. Ten WT-1-leukemias, were used as controls, andincluded 3 B-cell precursor ALLs and 7 AMLs.

All EBV BLCL and leukemia cells were typed for HLA A, B, C, DR and DQalleles at high resolution by standard techniques.

Assessment of T-cell Response.

IFNγ Production by WT-1 Specific T-Cells.

The proportion and phenotype (CD4 and CD8) of T-cells generating IFN′ inresponse to secondary stimulation with the WT-1 total pool, WT-1subpools or single WT-1 15-mer or 9-mer WT-1 peptides loaded onautologous PBMC were measured by FACS analysis of T-cells containingintracellular IFN′ as previously described (38,40).

Mapping of Immunogenic Epitopes.

Aliquots of the T-cells stimulated with the WT-1 total pool for 35-42days were washed and re-stimulated overnight with autologous PBMC loadedwith one of each of the subpools of WT-1 pentadeca peptides. T-cellresponses to each subpool were quantitated by FACS analysis of T-cellsbearing intracellular IFNγ as previously described (41). The mappinggrid (FIG. 2B) was then used to identify specific WT-1 15-mers uniquelyshared by 2 subpools eliciting T-cell responses. These 15-mers and 9-meror 11-mer sequences within the 15-mers were then analyzed as secondarysingle peptide stimulators to confirm their immunogenicity and definethe immunogenic epitope(s) within the 15-mer eliciting responses.

Cytotoxic Activity.

The W-1-specific and HLA-restricted cytotoxic activity of sensitizedT-cells was measured in standard Cr51 release assays against a panel ofHLA-matched and mismatched CAM targets either unmodified or loaded withthe total pool, the identified 15-mer, or the 9-mer or 11-mer epitope ofWT-1 eliciting T-cell responses, as previously described (32). Inaddition, the restricting HLA allele presenting each immunogenic epitopewas identified by measuring the cytotoxicity of the sensitized T-cellsagainst a panel of allogeneic CAMs pre-loaded with the peptide, eachsharing a single HLA allele expressed on the responding WT-1-specificT-cells as previously described (41). The cytotoxic activity of the WT-1epitope-specific CTLs against WT-1- and WT-1+ leukemia cell lines orprimary leukemic cells expressing the restricting HLA alleles was alsoassessed in this cytotoxicity assay Cr51 assay as previously described(32).

Immunogenicity of the Identified Immunodominant WT-1 Derived Epitopes.

To estimate the immunogenicity of identified WT-1 peptide epitopes indifferent individuals, enriched T-cells separated from PBMC of groups ofnormal donors expressing one of a series of prevalent HLA alleles (i.e.HLA-A0201, A0301, A2402, B0702) which were previously identified as apresenter of a newly identified WT-1 epitope were sensitized in vitrowith artificial antigen-presenting cells (AAPC) (42) expressing that HLAallele and loaded with the pre-identified WT-1 epitope or an irrelevantpeptide. The panel of AAPCs includes AAPCs expressing one of thefollowing single HLA alleles: HLA A0201, A0101, A0301, A2402, B0702 orB0801, which were generated as previously described (42). After 35 daysof co-culture of T-cells with the peptide-loaded AAPCs in the presenceof IL2, CTLs were secondarily stimulated overnight with autologous PBMCloaded with the sensitizing peptide or an unrelated peptide and testedfor their IFNγ response. The responses were registered as positive ifthe proportion of T-cells producing IFNγ in response to the secondarystimulation with autologous PBMC loaded with the stimulating WT-1derived peptide exceeded the background proportion of IFNγ T-cellsincubated with PBMC alone by two fold or more.

Example 2 Responses of Normal Donors to the WT-1 Total Pool ofPentadecapeptides

Frequencies of WT-1-specific IFNγ+ T-cells in the PBMC of 41 normaldonors were measured initially. These frequencies ranged between 0.01%to 1.82%, and exceeded the background of IFNγ+ T-cells detected inT-cells stimulated with autologous PBMC alone in only 10/41 individuals(FIG. 1A). In vitro sensitization of T-cells from 56 normal donors withautologous CAMs loaded with the total pool of WT-1 pentadecapeptides forperiods of 35-42 days resulted in significant expansion of IFNγ+ T-cellsin 41/56 cases (73%) (FIG. 1A). T-cells generated from 38/56 donors alsoexhibited cytotoxic activity against autologous PHA blasts loaded withthe WT-1 total pool (FIG. 1B), including T-cells from 38 of the 41donors that produced IFNγ in response to secondary stimulation with theWT-1 peptide pool.

The capacity of one of the previously reported WT-1 epitopes predictedto bind the HLA-A0201 allele, ₁₂₆₋₁₃₄RMFPNAPYL (SEQ ID NO:21; RMF) (43)were compared with the total pool of WT-1 pentadecapeptides to stimulateWT-1 reactive T-cells in HLA-A0201+ normal donors (n=14) when loaded onautologous CAMs. Increased frequencies of IFNγ+ T-cells initiallysensitized with the RMF peptide were detected in 9/14 donors, 7 of whomalso responded to secondary simulation with the pooled peptides (FIG.1C). In contrast, 12/14 CTL lines initially sensitized with the WT-1peptide pool, generated high frequencies of IFNγ+ T-cells aftersecondary stimulation with the WT-1 total pool, including 6 CTL linesthat also responded to RMF. The epitopes of WT-1 recognized by theT-cells sensitized with the total pool (vide infra) were mapped andepitopes other than RMF in 12/14 donors were identified. The magnitudeof the responses to those epitopes was much higher than to the RMFpeptide (FIG. 1C). Only 4/14 CTL lines initially sensitized with RMFexhibited cytotoxic activity against RMF-loaded autologous PHA blasts;of which 3 could also lyse autologous PHA blasts loaded with the WT-1pool (FIG. 1D). In contrast, 10/14 CTL sensitized with the pool of WT-1peptides were cytotoxic against PHA blasts loaded with the WT-1 totalpool including 3/14 that lysed RMF peptide loaded blasts (FIG. 1D).Thus, in a high proportion of HLA A0201+ donors, stimulation of T-cellswith the WT-1 total pool more consistently elicited WT-1-specific T-cellresponses than stimulation with the single HLA A0201 binding RMFpeptide.

Detailed description of FIG. 1. WT-1 specific responses of CTL generatedfrom PBMC of normal donors (n=56) by stimulation with autologous APCsloaded with total pool of WT-1 derived pentadecapeptides: A. productionof IFNγ in PBMC alone (as a background), PBMC co-incubated overnightwith the total pool of pentadecapeptides spanning the whole sequence ofWT-1 protein (PBMC+WT-1 pool) and pre-generated WT-1 specific T cellsco-incubated overnight with WT-1 peptide loaded PBMC; B. cytotoxicactivity of the WT-1 specific CTLs generated in vitro by stimulationwith WT-1 total pool against WT-1-(autologous PHA stimulated blasts) andWT-1+(autologous PHA stimulated blasts loaded with the total pool ofWT-1 pentadecapeptides) targets at 50:1 effector: stimulator ratio; C.IFNγ response measured by FACS staining in different responder cellpopulations (peripheral blood derived PBMC, pre-generated CTLssensitized in vitro with the RMF peptide loaded on autologous CAM andpre-generated CTLs sensitized with the total pool of WT-1 15-mers) aftersecondary overnight stimulation with autologous PBMC either unmodifiedor loaded with one of the following: RMF peptide, dominant epitopes ofWT-1 identified by the epitope mapping approach in the WT-1-total poolsensitized CTL, WT-1 total pool of the 141 pentadecapeptides; D.Cytotoxic activity of the WT specific T cells generated in vitro bysensitization with autologous CAMs loaded with the RMF 9-mer or with thetotal pool of the WT-1 15-mers. The cytotoxicity of the T cells wasassessed against autologous WT-1 negative targets (PHA activated blasts)and the same targets loaded with RMF peptide, the total pool of WT-115-mers or the dominant WT-1 epitope identified for the same T cellline.

Example 3 Identification of Immunogenic Epitopes of WT-1 ProteinRecognized by the WT-1-Reactive T-Cells

WT-1 CTLs Generated by Sensitization with the Pooled Peptides areEpitope Specific and HLA Restricted.

The epitopes recognized by T-cells sensitized in vitro with the totalpool of overlapping WT-1 pentadecapeptides (FIG. 2A) were identified byquantitating IFNγ+ T-cells responding to a mapping grid of subpools ofWT-1 15-mers formed so that any single 15-mer is shared by only 2intersecting subpools (FIG. 2B). As shown for a representative examplein FIG. 2C, significantly increased numbers of IFNγ+ T-cells areselectively generated in response to subpools #3 and #19 which share thepentadecapeptide #75. The T-cells were then stimulated with neighboring15-mers, each overlapping peptide #75 by 11aa. As can be seen, IFNγ+T-cells are selectively generated in response to peptide #75 (FIG. 2D).The newly identified immunogenic WT-1 epitope is ₁₇₄₋₁₈₂HSFKHEDPM.Subsequently, the cytotoxic activity of these T-cells was assessedagainst a panel of allogeneic CAMs either unmodified or loaded with thispeptide, each sharing one HLA allele expressed by the tested CTLs. Asshown in FIG. 2E, the T-cells selectively lysed peptide loadedautologous targets and targets expressing the HLA-B3501 allele, and didnot lyse peptide-loaded targets sharing other HLA alleles inherited bythe T-cell donor. These T-cells also lysed WT-1+BALL cells coexpressingthe HLA-B3501 allele.

Detailed description of FIG. 2. Strategy for the generation of the totalpool of overlapping pentadecapeptides spanning the whole sequence of theWT-1 protein and epitope mapping: A. The sequence of the WT-1 proteinconsisting of 575 amino acids and the principle of 11 amino acidoverlapping pentadecapeptides are illustrated. A total of 141pentadecapeptides are required to span the entire protein. The sequenceof 575 aminoacids published by Gessler et al. (37), was employed. Thissequence includes an additional 126 aminoacids in the N-terminus. Inorder to match the sequential numbers of aminoacids within the WT-1sequence used with the longest, most frequently described WT-1 isoform Dwe numbered the first 126aa with negative values and used the positivevalues to number the subsequent 449 aminoacids described in the longestisoform D; B. The mapping grid consisting of 24 subpools each containingup to 12 WT-1-derived pentadecapeptides. Each peptide is uniquelycontained within two intersecting subpools: for example peptide 75 isuniquely shared by subpools 3 and 19; C. IFNγ production by WT-1sensitized CTLs in response to secondary overnight stimulation with thesubpools of WT-1 pentadecapeptides loaded on autologous PBMC. Dominantresponses are observed for the subpools #3 and #19 both containing onecommon pentadecapeptide #75; D. IFN′ production by the WT-1 CTLs inresponse to secondary overnight stimulation with the singlepentadecapeptide contained within the subpools eliciting the highestresponses as per the analysis determined in 2C of this figure confirmsthat the dominant immunogenic sequence is contained withinpentadecapeptide #75; E. HLA restriction of the WT-1 specific T cellsresponding to peptide #75 identified by Cr51 release assay against apanel of allogeneic CAMs or PHA blasts matching single HLA allelesexpressed by the WT-1CTL donors. These are presented along the X axis ofthe graph. The CAMs or PHA blasts used in the assay are unmodified (greybars) or loaded with the WT-1 dominant epitope (black bars). The WT-1specific cytotoxic activity of the WT-1 CTLs is restricted by the B3501HLA allele.

Mapping of WT-1 Peptides Eliciting T-Cell Responses Identifies aDiversity of Immunogenic Epitopes Presented by Different Class I and IIHLA Alleles.

The same approach was used to map and ultimately identify WT-1 epitopeseliciting responses by T-cells from the other 40 responding normaldonors. Of these donors, 8 (19%) responded exclusively to one WT-1peptide, while 18 (43%) responded to two and 16 (39%) to 3 peptides. Incultures eliciting responses to more than one WT-1 peptide, the patternsof IFNγ+ T-cell responses to the subpools were sufficiently distinctiveto permit initial segregation of potentially immunogenic peptides. Eachcandidate peptide was then evaluated individually to ascertain thespecific peptide inducing a T-cell response.

The immunogenic peptides of WT-1 that were identified and theirpresenting HLA alleles are listed in Table 1. Of the 42 WT-1 peptideseliciting T-cell responses, 41 are newly identified; only one of theseWT-1 peptides, the ₁₂₆₋₁₃₄RMFPNAPYL nonamer presented by HLA-A0201, hasbeen previously described and shown to be immunogenic when presented bythis allele (43) Peptide 91, ₂₃₅₋₂₄₉CMTWNQMNLGATLKG contains an epitopewhich, in the study, elicited CD4+ T-cell responses restricted by HLADRB 1 0402, but also contains the ₂₃₅₋₂₄₃ CMT nonamer known to bepresented by HLA A0201 and HLA A2402 (29). For 26 of the peptidespresented by class I HLA alleles, a single presenting HLA allele wasidentified in the initially studied donor. However, when theHLA-restrictions of T-cells responding to these peptides in differentdonors was examined, 10 of these peptides were found that could elicitT-cell responses when presented by 2 or 3 different class I HLA alleles.One sequence, the ₂₃₈₋₂₄₆WNQMNLGAT peptide, elicited strong IFNγ+ CD8+T-cell responses when presented in different donors by any one of 4distinct HLA class I alleles.

TABLE 1 WT-1 derived immunogenic epitopes identified by IFNγ productionassay for T cells responses using pool of overlapping pentadecapeptidesspanning the whole sequence of WT-1protein. Shaded rows represent peptides thatcan be presented by more than one HLA allele. Bolded peptide sequencesrepresent those tested in Example 5 and results shown in Table 3.Cytotoxic CTL response, 15-mer %(at 50:1) E:T ratio vs numberIFNg response of WT-1 (SEQ ID, cells, % IFNg + peptide Table IV) cellsWT-1- loaded Containing the Present- WT-1 Auto- auto- WT-1− WT-1+dominant Sequence  ing HLA No WT-1 peptide logous logous leuke leuke-epitope identified allele peptide loaded APC APC mia mia  #1(-125)-(-117) B0702 0.9 11.3 0 27 1 67 RQRPHPGAL (SEQ ID NO: 142)  #2(-119)-(-111) B0702 0.5 14.0 0 30 1 60 GALRNPTAC (SEQ ID NO: 143)  #4(-110)-(-102) A0201 0.98 5.75 0 30 2 22 PLPHFPPSL (SEQ ID NO: 144)  #5(-107)-(-99) A3101 0.73 4.82 0 42 ND ND HFPPSLPPT (SEQ ID NO: 145)  #7(-99)-(- B4001 1.5 12.8 0 45 3 65 91)THSPTHPPR A0201 0.4 5 2 50 0 38(SEQ IL) NO: 146) #13 (-75)-(-67) A0201 0.61 5.07 0 18 3 19 AILDFLLLQ(SEQ ID NO: 147) #20 (-47)-(-39) A0201 0.2 3.67 6 54 5 19 PGCLQQPEQB4701 0.5 4.6 6 54 ND ND (SEQ ID NO: 148) (-47)-(-37) DRB101 0.33 3.1 654 ND ND PGCLQQPEQQG 01 (SEQ ID NO: 149) #24-25  (-27)-(-19) A0201 1.054.48 3 41 10 37 KLGAAEASA (SEQ ID NO: 150) #29-30  (-8)-(1) B3501 0.071.0 5 73 5 39 ASGSEPQQM (SEQ ID NO: 151) #33 6-15 A0201 1.1 11.0 2 51 09 RDLNALLPAV** B5701 0.19 1.24 3 44 ND ND (SEQ ID NO: 152) #37 22-31A0201 0.07 0.9 8 32 3 47 GGCALPVSGA (SEQ ID NO: 153) #39 30-38 B3901 0.11.3 2 31 ND ND GAAQWAPVL (SEQ ID NO: 154) #41 38-46 A0201 0.2 4.18 0 730 40 LDFAPPGAS (SEQ ID NO: 155) 38-48LDFAPPGASAY DRB104 0.2 1.41 0 73 040 (SEQ ID NO: 156) 02 #43 46-54SAYGSIGGP A0201 1.2 6.46 2 51 0 0(SEQ ID NO: 157)* B4001 1.09 6.84 2 41 3 68 #46 58-66 A0201 1.15 6.69 240 0 0 PAPPPPPPP** (SEQ ID NO: 158) #58 106-114 B4402 0.92 5.65 8 46 NDND ACRYGPFGP (SEQ ID NO: 159) #62 122-130 B3503 0.78 2.0 0 84 ND NDSGQARMFPN* (SEQ ID NO: 160) C0401 0.78 2.0 0 84 ND ND #62-63  126-134A0201 0.52 2.17 3 41 2 25 RMFPNAPYL* (SEQ ID NO: 161) #65-66  135-143B3501 0.07 0.61 0 35 ND ND PSCLESQPA (SEQ ID NO: 162) #68 146-154 A01010.92 4.0 2 19 ND ND NQGYSTVTF (SEQ ID NO: 163) #73 166-174 B3801 0.813.14 0 26 ND ND HHAAQFPNH (SEQ ID NO: 164) #74-75  174-182 B3501 1.318.0 0 50 5 45 HSFKHEDPM (SEQ ID NO: 165) #82 202-210 B4402 1.02 3.77 837 ND ND CHTPTDSCT (SEQ ID NO: 166) #83-84  209-217 A0101 0.03 0.29 0 213 33 CTGSQALLL (SEQ ID NO: 167) #83 206-214 B3802 0.71 4.02 0 88 ND NDTDSCTGSQA (SEQ ID NO: 168) B4402 1.01 4.2 1 36 1 56 #86 218-226 B35030.84 3.0 0 84 4 48 RTPYSSDNL*** (SEQ ID NO: 169) C0401 0.84 3.0 0 84 448 #87 225-233 A0201 0.13 0.9 3 87 0 0 NLYQMTSQLE** (SEQ ID NO: 170) #91238-246 A0201 1.34 8.0 0 18 1 19 WNQMNLGAT C1701 2.1 12.0 0 10 1 16(SEQ ID NO: 171) A0101 2.1 7.31 0 26 ND ND B3508 1.23 5.0 0 18 4 19#91-92  239-248 A2402 0.02 0.14 4 9 1 17 NQMNLGATL (SEQ ID NO: 172) #91238-248 DRB11 0.59 6.0 0 8 0 0 WNQMNLGATLK 04 (SEQ ID NO: 173) DRB1040.07 0.53 4 16 1 17 235-249 02 CMTWNQWNLGAT LKG (SEQ ID NO: 174) #92242-250 A0101 0.32 1.83 2 19 ND ND NLGATLKGV A0201 0.06 0.75 1 18 2 19(SEQ ID NO: 175) #92-93  243-252 A0203 0.54 2.1 0 35 ND ND LGATLKGVAA(SEQ ID NO: 176) #93 246-253 0.09 1.85 4 80 ND ND TLGVAAGS A6901(SEQ ID NO: 177) #99-100 269-278 A0101 0.12 2.43 0 27 0 33 GYESDNHTTB3501 0.1 0.61 0 35 ND ND (SEQ ID NO: 178) 112-113 323-332 B3501 1.318.0 0 70 5 45 FMCAYPGCNK DRB104 0.91 3.48 9 5 5 (SEQ ID NO: 179) 01320-334 KRPFMCAYPGC (SEQ ID NO: 180) #129  390-398 A0201 1.08 5.81 3 40ND ND RKFSRSDHL (SEQ ID NO: 181) #131  398-406 A0201 1.56 14.0 0 38 NDND LKTHTTRTHT (SEQ ID NO: 182) #141  436-445 A0201 1.78 6.69 2 40 0 0NMHQRNFITKL** B4001 2.1 7.71 0 31 3 72 (SEQ ID NO: 183) A2402 0.61 2.7919 47 0 0 *-the epitope previously predicted by the computer algorithmor described in the literature **-T-cells cytotoxic against autologousWT-1 peptide loaded APCs but not leukemia cells ***-assignment of HLArestriction to one or other allele cannot be made due to lack of targetsinheriting one allele without the other

Using this epitope mapping strategy, 5 new 11-mer peptides wereidentified that stimulated CD4+ T-cell responses restricted by HLA classII alleles. The CD4+ T-cells generated in response to each of theseepitopes expressed high levels of IFNγ+ T-cells. The CD4+ T-cellsresponding to 3 of these 5 peptide epitopes also exhibited specificcytotoxic activity against peptide loaded PHA blasts as well asunmodified WT-1+ leukemic blasts selectively sharing the restrictingclass II HLA allele.

In 4 of the 56 donors tested, epitope mapping of T-cells sensitized withthe complete pool of WT-1 15-mers identified specific 15-mers elicitingboth CD4+ and CD8+ T-cell responses (15-mer peptides #20, 41, 91, 112).Fine mapping of the sequences eliciting these responses identified four11-mers that stimulated HLA class II-restricted CD4+ T-cell responseswhich also contained, within their sequences, 9-mers that elicited HLAclass I-restricted CD8+ T-cell responses. A representative example ofone of these dual stimulating peptides is presented in FIG. 3. In thiscase, peptide 41 was found to elicit both CD4+ and CD8+IFNγ+ T-cellresponses (FIG. 3A). Fine mapping of the 11-mers within peptide 41eliciting the CD4+ IFNγ+ T-cell response (FIG. 3A) suggested the₃₈₋₄₈LDFAPPGASAY peptide as the most immunogenic sequence inducing bothCD4+ and CD8+IFNγ+ T-cell responses. Strikingly, the peptide 41sensitized T-cells lysed PHA blasts sensitized with either the 9aasequence (₃₈₋₄₆LDFAPPGAS) or the 11aa sequence (₃₈₋₄₈LDFAPPGASAY), butdid not lyse PHA blasts loaded with the ₃₆₋₄₆PVLDFAPPGAS or₃₇₋₄₇VLDFAPPGASA 11-mers. Subsequent examination of the HLA restrictionof the T-cells in the culture (FIG. 3D) revealed that the class IIHLA-restricted T-cells were selectively cytotoxic against targetssharing the alleles DRB 1 0402 and DQB 1 0302 only when loaded with theLDF 11-mer, while the T-cells restricted by HLA A0201 were able to lysetargets loaded with either the 11-mer or the 9-mer LDF peptide. In thiscase, it was not possible to ascertain whether DRB1 0402 or DQB 1 0302was the restricting class II HLA allele because cells were not availablein the panel expressing one without the other.

Detailed description of FIG. 3. HLA class I and II restricted WT-1specific T cell respond to the same immunodominant peptide 15-merderived from WT-1 protein in the WT-1 CTL sensitized with the WT-1 totalpool of overlapping 15-mers loaded on autologous CAMs. A. Production ofIFNg by the CD8+ and CD4+WT-1 specific T cells in response to secondaryovernight stimulation with the same dominant WT-1 derived 15-mer #41; B.Identification of the immunogenic sequence of aminoacids withinpentadecapeptide #41 by IFNg production after secondary overnightstimulation with autologous PBMC loaded with a panel of 9-mers eitherunique for the peptide #41 (LDF—LDFAAPGAS) or contained within theneighboring overlapping 15-mer #40 (PVL—PVLDFAPPG, VLD—VLDFAPPGA) and#42 (DFA—DFAPPGASA). Only the 9-mer uniquely presented within the 15-mer#41, LDF, elicits an IFNg response; C. Peptide-specific cytotoxicactivity of WT-1 CTL against the panel of 9-mers and 11-mers containedwithin peptide #41 and loaded on autologous PHA stimulated blasts isobserved against both the 11-mer LDF and 9-mer LDF contained within the11-mer LDF as determined in a standard Cr51 release assay at 25:1 E:Tratio; D. HLA restriction of the cytotoxic activity of the WT-1 CTL:T-cells restricted by HLA-A0201 lyse targets loaded with either the11-mer or the 9-mer, while those restricted by HLA DRB 10402 only lysedtargets loaded with the 11-mer.

Example 4 T-Cells Generated Against Newly Identified WT-1 EpitopesExhibit Cytotoxic Activity Against WT-1+ Leukemias

Once the WT-1 peptide specificity was established and HLA restrictionsof the IFNγ+ T-cells responding to the pool of WT-1 peptides, theircytotoxic activity was examined against unmodified and peptide loadedautologous PHA blasts and against a series of allogeneic PHA blastsloaded with the identified peptides as well as primary acute leukemiccell blasts expressing WT-1 protein that coexpressed the WT-1 specificT-cells' restricting HLA allele. For the latter tests, WT-1+ leukemiccells not expressing the restricting allele and WT-1-cells sharing therestricting allele served as controls. Results are summarized in Tables1 and 2.

As can be seen in Table 1, of 51 cultures generating IFNγ+CD8+ T-cellsafter secondary stimulation with an identified peptide loaded autologousAPC, 50 also exhibited significant specific cytotoxic activity againstautologous PHA blasts loaded with the targeted peptide. Of these, 48also lysed allogeneic peptide loaded PHA blasts or DCs sharing therestricting HLA allele of the responding T-cells. CD4+ IFNγ+ T-cellsresponding to 3/5 identified 11-mer peptides presented by class II HLAalleles also lysed peptide loaded autologous and HLA-sharing allogeneicclass II+ targets.

Of the T-cell cultures exhibiting epitope-specific cytotoxic activityagainst peptide loaded targets, 36 could be tested for cytotoxicactivity against WT-1+ leukemic cells coexpressing the T-cell'srestricting HLA allele. Of these 36, 27 exhibited HLA-restrictedcytotoxic activity against the WT-1+ leukemic cells (Table 2). T-cellsspecific for five peptides, ₆₋₁₅RDL, ₄₆₋₅₄SAY, ₅₈₋₆₆PAP, ₂₂₅₋₂₃₃NLY, and₄₃₆₋₄₄₅NMH, presented by HLA A0201, could not lyse HLA-A0201⁺ WT-1+leukemic cells. However, HLA B4001 restricted T-cells specific for the₄₆₋₅₄SAY peptide, could lyse WT-1+ leukemic coexpressing this HLAallele. Similarly, NMH peptide-specific HLA-restricted T-cell lines thatlysed targets loaded with the NMH peptide coexpressing HLA A0201, B4001or A2402 were only able to lyse

WT-1+ leukemic cells expressing the HLA B4001 allele.

TABLE 2 WT-1 derived immunogenic epitopes identified by IFNγ productionassay for T cells responses using pool of overlapping pentadecapeptides spanning the whole sequence of WT-1 protein. Bold sequences indicate peptides tested as described in Example 5 and results provided in Table 3. Cytotoxic CTL response, % (at 50:1)E:T ratio vs WT-1− WT-1+ allo Prediction algorithm allo APCAPC with Dis- with restricting associa- restrict- HLA allele PresentingSequence Binding tion ing HLA loaded with WT-1 WT-1+ HLA alleleidentified index time allele WT-1 peptide leukemia leukemia A0101146-154 3 0.001 4 15 ND ND NQGYSTVTF SEQ ID NO: 163 209-217 12 0.125 026 3 33 CTGSQALLL SEQ ID NO: 167 238-246 2 0 3 19 ND ND WNQMNLGATSEQ ID NO: 171 242-250 3 0.01 1 17 ND ND NLGATLKGV SEQ ID NO: 175269-278 15 1.5 0 26 0 33 GYESDNHTT SEQ ID NO: 178 323-332 0 0.1 2 0 5 0FMCAYPGCNK** SEQ ID NO: 179 A0201 (-110)-(-102) 21 2 1 24 2 22 PLPHFPPSLSEQ ID NO: 144 (-99)-(-91) 3 0 1 21 0 38 THSPTHPPR SEQ ID NO: 146(-75)-(-67) 19 0.272 3 17 3 19 AILDFLLLQ SEQ ID NO: 147 (-47)-(-39) 0 07 27 5 19 PGCLQQPEQ SEQ ID NO: 148 (-27)-(-19) 19 17 2 22 10 37KLGAAEASA SEQ ID NO: 150 6-15 18 0.2 4 31 0 9 RDLNALLPAV SEQ ID NO: 15222-31 13 0.003 3 25 3 47 GGCALPVSGA SEQ ID NO: 153 38-46 11 0 1 62 0 40LDFAPPGAS SEQ ID NO: 155 46-54 14 0 5 31 0 0 SAYGSLGGP** SEQ ID NO: 15758-66 5 0 1 18 0 0 PAPPPPPPP** SEQ ID NO: 158 126-134 22 313 1 52 2 25RMFPNAPYL* SEQ ID NO: 161 225-233 23 68 3 28 0 0 NLYQMTSQLE**SEQ ID NO: 170 238-246 19 0.3 0 21 1 19 WNQMNLGAT SEQ ID NO: 171 242-25024 160 1 14 2 19 NLGATLKGV SEQ ID NO: 175 390-398 11 0.054 1 27 ND NDRKFSRSDHL SEQ ID NO: 181 398-406 5 0.18 1 22 ND ND LKTHTTRTHTSEQ ID NO: 182 436-445 20 15 4 32 0 0 NMHQRNHTKL** SEQ ID NO: 183 A0203243-252 19 NA 0 21 ND ND LGATLKGVAA SEQ ID NO: 176 A2402 239-248 10 7.20 2 1 17 NQMNLGATL SEQ ID NO: 172 436-445 13 0.6 13 27 0 0 NMHQRNHTKL**SEQ ID NO: 183 A6901 246-253 NA NA 0 57 ND ND TLGVAAGS SEQ ID NO: 177B0702 (-125)-(-117) 15 40 1 53 1 67 RQRPHPGAL SEQ ID NO: 142(-119)-(-111) 2 0.3 5 22 1 60 GALRNPTAC SEQ ID NO: 143 A3101(-107)-(-99) NA 0.01 0 27 ND ND HFPPSLPPT SEQ ID NO: 145 B3501 (-8)-(-1)NA 15 3 51 5 39 ASGSEPQQM SEQ ID NO: 151 135-143 NA 0.075 0 21 ND NDPSCLESQPA SEQ ID NO: 162 174-182 NA 10 3 63 5 45 HSFKHEDPMSEQ ID NO: 165 269-278 NA 0.004 0 23 ND ND GYESDNHTT SEQ ID NO: 178323-332 NA 0.01 0 61 5 45 FMCAYPGCNK SEQ ID NO: 179 B3503 122-130 NA NA3 41 ND ND SGQARMFPN SEQ ID NO: 160 218-226 NA NA 3 31 4 48 RTPYSSDNLSEQ ID NO: 169 B3508 238-246 NA NA 2 21 4 19 WNQMNLGAT SEQ ID NO: 171B3802 206-214 NA NA 1 53 ND ND TDSCTGSQA SEQ ID NO: 168 B3801 166-174 110.3 1 17 ND ND HHAAQFPNH SEQ ID NO: 164 B3901 30-38 12 3 0 19 ND NDGAAQWAPVL SEQ ID NO: 154 B4001 (-99)-(-91) 3 0.02 0 31 3 65 THSPTHPPRSEQ ID NO: 146 46-54 1 0.002 8 24 3 68 SAYGSLGGP SEQ ID NO: 157 436-4451 0.002 1 26 3 72 NMHQRNHTKL SEQ ID NO: 183 B4402 202-210 3 NA 7 19 NDND CHTPTDSCT SEQ ID NO: 166 206-214 2 NA 0 88 1 56 TDSCTGSQASEQ ID NO: 168 106-114 4 NA 7 23 ND ND ACRYGPFGP SEQ ID NO: 159 B4701(-47)-(-37) 1 NA 1 25 ND ND PGCLQQPEQ SEQ ID NO: 148 B5701 6-15 NA NA 122 ND ND RDLNALLPAV SEQ ID NO: 152 C0401 122-130 NA NA 3 41 ND NDSGQARMFPN SEQ ID NO: 160 C1701 238-246 NA NA 0 7 1 16 WNQMNLGATSEQ ID NO: 171 DRB10101 (-47)-(-37) 8 NA 1 25 ND ND PGCLQQPEQQGSEQ ID NO: 149 DRB10402 38-48 NA NA 1 71 0 40 LDFAPPGASAY SEQ ID NO: 156DRB10402 235-249 NA NA 2 15 1 17 CMTVVNQMNLGA TLKG SEQ ID NO: 174DRB10401 320-334 22 NA 3 0 5 5 KRPFMCAYPGC SEQ ID NO: 180 DRB11104238-248 NA NA 2 1 0 0 WNQNINLGATLK SEQ ID NO: 173 *-previously reportedepitopes; **-T cells cytotoxic against the autologous WT-1 peptideloaded APC but not the leukemic cells.

To ascertain that the cytotoxic activity of the WT-1 peptide-specificT-cells observed against allogeneic WT-1+ leukemic cells sharing theT-cells restricting allele does not reflect the presence ofalloresponsive T-cells in the T-cell lines, we tested the cytotoxicactivity of 13 of these HLA-restricted WT-1 peptide specific T-celllines against WT-1+ leukemic cells and WT-1-PHA blasts cultured from thesame leukemic patient. As shown in Table 3a, the WT-1 specific T-cellslysed the WT-1+ leukemic cells but not PHA blasts from the same patient.

TABLE 3a Cytotoxic activity of the T cells specific for WT-1derivedimmunogenic epitopes identified by IFNγ production assay forT cells responses using pool of overlapping pentadecapeptides spanning the whole sequence of WT-1 protein and tested against WT-1 positive primary leukemic cellsand PHA blasts of the same origin.Cytotoxic CTL response, 15-mer % (at 50:1) E:T number ratio vsContaining Presenting  WT-1⁺ PHA the dominant HLA Leukemia blastsepitope Sequence identified allele ** *** #1 ⁽⁻¹²⁵⁾⁻⁽⁻¹¹⁷⁾RQRPHPGALB0702 67 2 SEQ ID NO: 142 #2 ⁽⁻¹¹⁹⁾⁻⁽⁻¹¹¹⁾ GALRNPTAC B0702 60 1SEQ ID NO: 143 #4 ⁽⁻¹¹⁰⁾⁻⁽⁻¹⁰²⁾PLPHFPPSL A0201 22 1 SEQ ID NO: 144 #7⁽⁻⁹⁹⁾⁻⁽⁻⁹¹⁾THSPTHPPR B4001 65 5 SEQ ID NO: 146 A0201 38 3 #24-25⁽⁻²⁷⁾⁻⁽⁻¹⁹⁾KLGAAEASA A0201 37 8 SEQ ID NO: 150 #29-30 ⁽⁻⁸⁾⁻⁽¹⁾ASGSEPQQMB3501 39 9 SEQ ID NO: 151 #37 ₂₂₋₃₁ GGCALPVSGA A0201 47 6 SEQ ID NO: 153#43 ₄₆₋₅₄SAYGSLGGP* B4001 68 3 SEQ ID NO: 157 #62-63 ₁₂₆₋₁₃₄RMFPNAPYL*A0201 25 3 SEQ ID NO: 161 #86 ₂₁₈₋₂₂₆RTPYSSDNL B3503 48 1 SEQ ID NO: 169C0401 48 1 #141  ₄₃₆₋₄₄₅NMHQRNHTKL* B4001 72 1 SEQ ID NO: 183 P < 0.001*-the epitope previously predicted by the computer algorithm ordescribed in the literature **-leukemia samples were presented either byimmortalized leukemia cell lines or by primary leukemia cells obtainedfrom patients with WT-1⁺ leukemia ***-PHA blasts were generated fromPBMC derived from the same patients as the WT-1⁺ primary leukemia

PHA blasts were not available from every patient that provided leukemiablasts for this study. Nevertheless, these results provide evidence thatthe cytotoxicity of the WT-1 specific T-cells is not ascribable tocontaminating alloreactivity. A second, more inclusive, but less directline of evidence is provided by a paired comparison of the responses ofT-cells derived from 35 of the donors that had been contemporaneouslysensitized in vitro against either WT-1 peptide pool loaded orunmodified autologous EBVBLCL, against these primary leukemias. As shownin Table 3b, T-cells sensitized with the WT-1 peptide pool-loadedEBVBLCL lysed WT-1+ leukemic cells sharing the T-cells' restricting HLAallele in 25 of 35 cases. In contrast, T-cells sensitized withautologous EBVBLCL alone consistently failed to lyse the same WT-1+leukemia targets.

TABLE 3b Leukemocidal activity of defined epitope-specific and HLArestricted T cells from normal donors sensitized with eitherautologous EBV BLCL or EBV BLCL loaded with pooled WT-1 peptides against primary WT-1⁺ leukemia sharing the Tcells restricting HLA alleles. 15-mer Cytotoxic CTL response, number%(at 50:1) E:T ratio vs Containing  Pre- WT-1⁺ leukemia the senting expressing restricting dominant HLA HLA allele epitopeSequence identified allele WT-1 CTL EBV CTL #1 ⁽⁻¹²⁵⁾⁻⁽⁻¹¹⁷⁾RQRPHPGALB0702 67 1 SEQ ID NO: 142 #2 ₍₋₁₁₉₎₋₍₋₁₁₁₎GALRNPTAC B0702 60 2SEQ ID NO: 143 #4 ⁽⁻¹¹⁰⁾⁻⁽⁻¹⁰²⁾PLPHFPPSL A0201 22 3 SEQ ID NO: 144 #7⁽⁻⁹⁹⁾⁻⁽⁻⁹¹⁾THSPTHPPR B4001 65 0 SEQ ID NO: 146 A0201 38 3 #13 ⁽⁻⁷⁵⁾⁻⁽⁶⁷⁾AILDFLLLQ A0201 19 5 SEQ ID NO: 142 #20 ⁽⁻⁴⁷⁾⁻⁽³⁹⁾ PGCLQQPEQ A0201 19 10SEQ ID NO: 1148 #24-25 ⁽⁻²⁷⁾⁻⁽⁻¹⁹⁾KLGAAEASA A0201 37 5 SEQ ID NO: 150#29-30 ⁽⁻⁸⁾⁻⁽¹⁾ASGSEPQQM B3501 39 0 SEQ ID NO: 151 #33 ₆₋₁₅ RDLNALLPAV**A0201 9 0 SEQ ID NO: 152 #37 ₂₂₋₃₁ GGCALPVSGA A0201 47 3 SEQ ID NO: 153#41 ₃₈₋₄₆ LDFAPPGAS A0201 40 0 SEQ ID NO: 1554 ₃₈₋₄₈LDFAPPGASAY DRB₁0440 0 SEQ ID NO: 156 02 #43 ₄₆₋₅₄SAYGSLGGP* A0201 0 0 SEQ ID NO: 157B4001 68 3 #46 ₅₈₋₆₆PAPPPPPPP* A0201 0 0 SEQ ID NO: 158 #62-63₁₂₆₋₁₃₄RMFPNAPYL* A0201 25 2 SEQ ID NO: 161 #74-75 ₁₇₄₋₁₈₂HSFKHEDPMB3501 45 5 SEQ ID NO: 165 #83-84 ₂₀₉₋₂₁₇CTGSQALLL A0101 33 3SEQ ID NO: 167 #83 ₂₀₆₋₂₁₄TDSCTGSQA B4402 56 1 SEQ ID NO: 168 #86₂₁₈₋₂₂₆RTPYSSDNL B3503 48 4 SEQ ID NO: 169 C0401 48 4 #87₂₂₅₋₂₃₃NLYQMTSQLE* A0201 0 0 SEQ ID NO: 170 #91 ₂₃₈₋₂₄₆ WNQMNLGAT A020119 1 SEQ ID NO: 171 C1701 16 1 B3508 19 4 #91-92 ₂₃₉₋₂₄₈NQMNLGATL A240217 1 SEQ ID NO: 172 #91 ₂₃₈₋₂₄₈WNQMNLGATLK DRB₁11 0 0 SEQ ID NO: 173 04₂₃₅₋₂₄₉CMTWNQMNLGATLKG DRB₁04 17 1 SEQ ID NO: 174 02 #92₂₄₂₋₂₅₀NLGATLKGV A0201 19 2 SEQ ID NO: 175  #99-100 ₂₆₉₋₂₇₈GYESDNHTTA0101 33 0 SEQ ID NO: 178 #112-113 ₃₂₃₋₃₃₂FMCAYPGCNK B3501 45 5SEQ ID NO: 179 ₃₂₀₋₃₃₄KRPFMCAYPGC DRB₁04 5 5 SEQ ID NO: 180 01 #141 ₄₃₆₋₄₄₅NMHQRNHTKL* A0201 0 0 SEQ ID NO: 183 B4001 72 3 A2402 0 0 p <0.001 *-the epitope previously predicted by the computer algorithm ordescribed in the literature

Example 5 Immunogenicity of the Newly Identified WT-1 Epitopes

In order to ascertain that that the peptides identified by mappingresponses in single donors were also immunogenic in a high proportion ofindividuals bearing the same presenting HLA allele, it was determinedwhether these epitopes could elicit appropriately restricted T-cellresponses in groups of 6-12 individuals expressing that HLA allele. Forthis purpose, the T-cells from each donor were sensitized with theidentified epitope loaded on a panel of artificial antigen presentingcells (AAPC) (42) each expressing a single HLA allele, specificallyA0201, A0301, A2402 or B0702. As shown in Table 4, of 9 peptidesidentified that are presented by HLA-A0201, all were able to stimulateWT-1-specific IFNγ+ T-cell responses in a proportion of HLA-A0201+individuals. The previously reported 126-134RMFPNAPYL peptide presentedby HLA-A0201 allele elicited responses in 5/12 (42%) HLA-A0201+ normaldonors tested. In comparison, 5 of the other 8 peptides tested elicitedWT-1 peptide-specific responses in 50-75% of the same HLA-A0201+ donors.Two WT-1 epitopes presented by the HLA-B0702 allele also elicited WT-1specific T-cell responses in 50% and 63% of the tested individualsrespectively (Table 4). All of the peptides tested elicited specificresponses in at least 2 additional donors bearing their presenting HLAallele.

TABLE 4Proportion of normal donors responding to indentifed WT-1 peptidesloaded on AAPCs expressing a single HLA allele. Sequence  Proportionpreviously Identified  of normal WT-1 sequence Proportion identified in # of  donors predicted to of HLA  to be donors respondingbeimmunogenic responses allele presented  after  to the when presentedin normal ex- by the total pool peptide Predicted by the Predicteddonors to pressed  HLA allele  stimula- loaded on Bind- Dissoc-HLA alleles Bind- Dissoc- the by expressed tion on AAPCs ing iationexpressed by  ing iation peptide AAPC by the AAPC CAMs (%) index timethe AAPC index time stimulation A0201 (-99)-(-91) 1 6/12 3 0 (−99)-(−91)3 0  6/12 THSPTHPPR (50%) THSPTHPPR (50%) SEQ ID NO: 146 SEQ ID NO: 146(-75)-(-67) 1 8/12 19 0.272 (-78)-(-70) 28 225  8/12 AILDFLLLQ (67%)LLAAILDFL (67%) SEQ ID NO: 147 SEQ ID NO: 184 (-47)-(-39) 2 2/12 0 0(-45)-(-36) 21 70  2/12 PGCLQQPEQ (16%) CLQQPEQQGV (16%) SEQ ID NO: 148SEQ ID NO: 185 (-27)-(-19) 1 8/12 19 17 (−27)-(−19) 19 17  8/12KLGAAEASA (67%) KLGAAEASA (67%) SEQ ID NO: 149 SEQ ID NO: 150 6-15 13/12 18 0.2 7-15 27 12  3/12 RDLNALLPAV (25%) DLNALLPAV (25%)SEQ ID NO: 152 SEQ ID NO: 186 10-18 33 181  3/12 ALLPAVPSL (25%)SEQ ID NO: 187 22-31 3 9/12 13 0.003 22-31 13 0.003  9/12 GGCALPVSGA(75%) GGCALPVSGA (75%) SEQ ID NO: 153 SEQ ID NO: 153 38-46 2 8/12 11 037-45 16 4  0/12 LDFAPPGAS (67%) VLDFAPPGA (0%) SEQ ID NO: 155SEQ ID NO: 188 126-134 1 5/12 22 313 126-134 22 313  5/12 RMFPNAPYL(42%) RMFPNAPYL (42%) SEQ ID NO: 161 SEQ ID NO: 161 238-246 2 3/12 190.3 235-243 17 1.5 0/8 WNQMNLGAT (25%) CMTWNQMNL SEQ ID NO: 171SEQ ID NO: 189 total pool 13/27 8/12 (48%) (67%) A0301 126-134 1 2/8  104.5 124-133 14 0.001 0/8 RMFPNAPYL (25%) QARMFPNAPY SEQ ID NO: 161SEQ ID NO: 190 total pool 1/8 2/8  (12%) (25%) A2402 239-248 1 4/6  107.2 235-243 10 4 1/6 NQMNLGATL (60%) CMTWNQMNL (17%) SEQ ID NO: 172SEQ ID NO: 189 total pool 2/6 6/6  (33%) (100%) B0702 (-125)-(-117) 14/8  15 40 (−125)-(−117) 15 40 4/8 RQRPHPGAL (50%) RQRPHPGAL (50%)SEQ ID NO: 142 SEQ ID NO: 142 (-119)-(-111) 1 5/8  2 0.3 (−118)-(−109)15 120 5/8 GALRNPTAC (63%) ALRNPTACPL (63%) SEQ ID NO: 143SEQ ID NO: 191 323-332 1 3/8  1 0.015 327-335 17 0.4  4/8 FMCAYPGCNK(38%) YPGCNKRYF (50%) SEQ ID NO: 179 SEQ ID NO: 192 Total pool  2/8 3/8 (25%) (38%) DRB1 38-48 1 0/2  NA NA 35-49 20 NA ND 0402 LDFAPPGASAY(tested on APVLDFAPPGAS SEQ ID NO: 156 CAMs not AYG on AAPC)SEQ ID NO: 193

Example 6 Comparison of Responses to Peptides Identified by MappingResponses to Pooled WT-1 15-Mers with Responses to Previously ReportedWT-1 Peptides Predicted by Binding Algorithms to be Immunogenic

Primary responses by normal donor T-cells were compared to individualWT-1 peptides identified by the mapping strategy to responses againstother WT-1 peptides containing flanking sequences predicted to have ahigher binding index for the presenting HLA allele using bindingalgorithms previously described (44,45). As shown in Table 4 above, thepredicted binding indices for 8/12 mapped epitopes were only somewhatlower than those for the most studied WT-1 peptide, RMF, presented byHLA A0201. However, their dissociation times were markedly lower.Nevertheless, T-cell responses to each of these peptides were elicitedin a high proportion of normal donors.

In five instances, the mapped peptide specificity (i.e. ⁽⁻⁹⁹⁾⁻⁽⁻⁹¹⁾THS,⁽²²⁾⁻⁽⁻¹⁹⁾KLG, ₂₂₋₃₁GGC, ₁₂₆₋₁₃₄RMF and ⁽⁻¹²⁵⁾⁻⁽⁻¹¹⁷⁾RQR) was identicalto the peptide with the highest affinity for the presenting HLA allelepredicted by the binding algorithm within the stimulating 15-mer. Inthose instances in which the mapped sequences and the sequencespredicted to have the highest binding index differed, the proportion ofdonors responding to individual mapped peptides were equal or greaterthan those generated in response to the neighboring epitopes predictedto have higher affinity. For example, IFNγ+ T-cell responses weregenerated to the ₃₈₋₄₆LDF peptide in 8/12 (67%) of HLA A0201 donorstested, while none responded to the predicted and previously reported(46) epitope ₃₇₋₄₅VLDFAPPGA. Similarly, among HLA A2402+ donors, 4/6donors (60%) responded to the ₂₃₉₋₂₄₈NQMNLGATL peptide while only ⅙responded to the ₂₃₅₋₂₄₃CMTWNQMNL peptide previously reported to bepresented by this allele (29).

To directly compare peptides presented by HLA A0201 that were identifiedby matrix mapping with flanking peptides with higher predicted bindingindices, the peptides, mixed at equal concentration, were loaded on HLAA0201+ AAPCs and used to sensitize T-cells from 8 of the HLA A0201+normal donors. After 35 days of sensitization, the T-cells were thenwashed and secondarily restimulated for 24 hours with aliquots ofirradiated autologous PBMC loaded with each individual peptide.Responding IFNγ+ T-cells were then quantitated by FACS. The results,presented in FIG. 5, demonstrate that although the 22-31GGC peptide hasthe lowest binding index and the shortest predicted dissociation time,it induced strong IFNγ+ T-cell responses in 7/8 donors. Furthermore,although 3/8 donors responded to the ₆₋₁₅RDL, ₁₀₋₁₈ALL and ₇₋₁₅DLNpeptides, ₆₋₁₅RDL peptides identified by response mapping elicitedhigher numbers of IFNγ+ T-cells. In comparisons of the ⁽⁻⁷⁵⁾⁻⁽⁻⁶⁷)AILDFLLLQ with flanking _((—78)-(−70))LLAAILDFL sequence, the AILpeptide elicited superior responses and in a higher proportion of donors(6/8 vs. 3/8 donors). Similarly, in comparisons of the mapped₃₈₋₄₆LDFAPPGAS peptide with the previously reported ₃₇₋₄₅VLDFAPPGApeptide (46) the LDF peptide induced strong responses in 5 of the 8donors while the VLD peptide induced low responses in only 2 of thesedonors.

Detailed description of FIG. 5. IFNγ+ T-cell responses to equimolarmixtures of 9-mer peptides identified by epitope mapping of in vitroresponses and peptides within the same 15-mer or adjacent overlapping15-mer peptides predicted to have higher binding affinity andimmunogenicity. A. Responses to a mixture of nonamers spanning aminoacids+2 to +31 including the ₆₋₁₅RDL and ₂₂₋₃₁GGC peptides to which HLAA0201+ donors responded in epitope mapping studies. B. Responses to thein vitro mapped ⁽⁻⁷⁵⁾⁻⁽⁻⁶⁷⁾AILDFLLLQ epitope and a flanking peptide⁽⁻⁷⁸⁾⁻⁽⁻⁷⁰⁾LLAAILDFL with higher predicted binding affinity. C.Responses to the in vitro mapped ₃₈₋₄₆LDFAPPGAS epitope and theoverlapping ₃₇₋₄₅VLDFAPPGA predicted to have higher binding affinity.

FIG. 5 presents maps of the WT-1 protein. FIG. 5C defines thelocalization of all previously reported antigenic epitopes presented byHLA class I and II alleles; FIG. 5D depicts the location of immunogenicpeptides identified in this report. As can be seen, the 11 epitopespreviously reported to be presented by class I and 10 presented by classII HLA alleles are principally clustered in sequences encoded by exons1, 7 and 10, while the epitopes recognized by normal T-cells sensitizedwith the WT-1 peptide pool are principally clustered in sequencesencoded by the first 5 exons. Thus, 26 of the new epitopes are includedin each of the four major isoforms of WT-1 resulting from splicevariants that do or do not include the 17 amino acid sequence (aas250-266) in exon 5 or the three amino acid sequence (₄₀₀₋₄₁₀KTS) betweenzinc fingers 3 and 4. While the epitopes are broadly distributed,clusters of epitopes were detected in the RNA recognition domain in exon1 and the activation domain (aa 181-250) (FIG. 5F) proximal to thespliced 17aa segment in exon 5. The latter area also contained thoseepitopes most frequently recognized by multiple donors (FIG. 5E). Ninenewly identified epitopes map to a 126 amino acid sequence at the Nterminus encoded by a segment of the WT-1 gene initially described byGessler et al³⁷ that is centromeric to exon 1 of the (Exon 5⁺, KTS⁺)isoform of WT-1 and includes the long isoform of WT-1 initiated at a CUGcodon upstream of the AUG initiator for exon 1.⁵⁰ Strikingly, each ofthe epitopes identified in this sequence elicits IFNγ⁺ T-cells that arecytolytic against leukemic blasts coexpressing WT-1 and the T-cells'restricting HLA allele.

Example 7 Inhibitory Effect of Peptides in Ovarian Carcinoma

The utility of peptides described herein in treating ovarian cancer wasevaluated in two studies. In the first study, the inhibitory effect onovarian tumor engraftment of T-cells specific for different WT-1peptides was evaluated by pre-incubating T cells at different doses withSKOV3-A2 ovarian carcinoma cells before injection into NOD/SCID mice. Tcell cultures specific for the following immunodominant epitopes wereprepared using methods described above: A0201 restricted WT-1 peptideLKTHTTRTHT (SEQ ID NO:182) specific T cells; A0301 restricted WT-1peptide RQRPHPGAL (SEQ ID NO:142) specific T cells, and A0201 restrictedWT-1 peptide HFPPSLPPT (SEQ ID NO:145) T cells. T cells to tumor cellratios tested were 50:1, 10:1, 5:1 and control (no T cells). Followingtumor injection, the tumor burden was monitored by bioluminescentimaging. For all three T cell lines at each dose, a significantreduction in tumor burden was observed over time vs. control.Furthermore, mouse survival was prolonged by pre-incubation of tumorcells with WT-1 peptide specific T cells. In control groups, all micewere dead by 70 days post tumor injection. Increased survival was seendose-responsively with the T cell: tumor cell dose, which for the 50:1dose for all T cell lines still had some animals alive at 96 days, andalso at 10:1 for the LKTHTTRTHT specific line.

In a second experiment, WT-1 peptide specific T cells were administeredintravenously to NOD/SCID mice bearing pre-established ovarian carcinomaSKOV3-A2 xenografts. T cell lines evaluated were: A0201 restricted WT-1peptide LKTHTTRTHT (SEQ ID NO:182) specific T cells and A0301 restrictedWT-1 peptide RQRPHPGAL (SEQ ID NO:142) specific T cells. Tumor burdenwas monitored by bioluminescence, tumor infiltration by human CD3+ cellsevaluated and survival recorded. In both cases, the WT-1 specific Tcells afforded reduced tumor burden vs. control, increased tumorinfiltration by human CD3+ cells and increased survival.

Example 8 Recognition of Epitopes by Leukemia Patient T Cells

A phase I clinical trial was conducted using transplant donor-derivedT-cells sensitized with the full pool of WT-1 derived pentadecapaptidesdescribed above, in the adoptive therapy of patients who have relapsedfollowing an allogeneic marrow transplant from a normal related orunrelated donor. The HLA restricting alleles and correspondingimmunodominant WT-1 epitopes are as follows: A0201, SEQ ID NO:147;A0203, SEQ ID NOs:176 and 183; B3503 and C0401, SEQ ID NOs:161 and 169;A6901, SEQ ID NO:177; A0201, SEQ ID NO:182; B4701 and DRB₁ 0102, SEQ IDNOs: 148 and 149; A3101, SEQ ID NO:145; B4402, SEQ ID NOs:158 and 166;B3503, SEQ ID NOs:146 and 162; DRB₁ 1104, SEQ ID NO:149. It is notedthat several of the immunodominant epitopes eliciting the WT-1 specificT-cells that were used were directed against epitopes in the N-terminalregion of the gene, upstream from exon 1, i.e., SEQ ID NOs:145, 147, 148and 149. Two of the donors responded to PGCLQQPEQQG, SEQ ID NO:149, andboth treated patients had temporary clearance of WT-1⁺ leukemic cellsfollowing adoptive transfer.

Example 9 Pentadecapeptides

The following pentadecapeptides were synthesized. H2N refers to theN-terminal end of the peptide, and —COOH the C-terminus

TABLE 5 Sequence of pentadecapeptides  SEQ ID NO: 1. H2N-SRQRP HPGAL RNPTA-COOH  SEQ ID NO: 2. H2N-PHPGA LRNPT ACPLP-COOH  SEQ ID NO: 3. H2N-ALRNP TACPL PHFPP-COOH  SEQ ID NO: 4. H2N-PTACP LPHFP PSLPP-COOH  SEQ ID NO: 5. H2N-PLPHF PPSLP PTHSP-COOH  SEQ ID NO: 6. H2N-FPPSL PPTHS PTHPP-COOH  SEQ ID NO: 7. H2N-LPPTH SPTHP PRAGT-COOH  SEQ ID NO: 8. H2N-HSPTH PPRAG TAAQA-COOH  SEQ ID NO: 9. H2N-HPPRA GTAAQ APGPR-COOH SEQ ID NO: 10. H2N-AGTAA QAPGP RRLLA-COOH SEQ ID NO: 11. H2N-AQAPG PRRLL AAILD-COOH SEQ ID NO: 12. H2N-GPRRL LAAIL DFLLL-COOH SEQ ID NO: 13. H2N-LLAAI LDFLL LQDPA-COOH SEQ ID NO: 14. H2N-ILDFL LLQDP ASTCV-COOH SEQ ID NO: 15. H2N-LLLQD PASTC VPEPA-COOH SEQ ID NO: 16. H2N-DPAST CVPEP ASQHT-COOH SEQ ID NO: 17. H2N-TCVPE PASQH TLRSG-COOH SEQ ID NO: 18. H2N-EPASQ HTLRS GPGCL-COOH SEQ ID NO: 19. H2N-QHTLR SGPGC LQQPE-COOH SEQ ID NO: 20. H2N-RSGPG CLQQP EQQGV-COOH SEQ ID NO: 21. H2N-GCLQQ PEQQG VRDPG-COOH SEQ ID NO: 22. H2N-QPEQQ GVRDP GGIWA-COOH SEQ ID NO: 23. H2N-QGVRD PGGIW AKLGA-COOH SEQ ID NO: 24. H2N-DPGGI WAKLG AAEAS-COOH SEQ ID NO: 25. H2N-IWAKL GAAEA SAERL-COOH SEQ ID NO: 26. H2N-LGAAE ASAER LQGRR-COOH SEQ ID NO: 27. H2N-EASAE RLQGR RSRGA-COOH SEQ ID NO: 28. H2N-ERLQG RRSRG ASGSE-COOH SEQ ID NO: 29. H2N-GRRSR GASGS EPQQM-COOH SEQ ID NO: 30. H2N-RGASG SEPQQ MGSDV-COOH SEQ ID NO: 31. H2N-GSEPQ QMGSD VRDLN-COOH SEQ ID NO: 32. H2N-QQMGS DVRDL NALLP-COOH SEQ ID NO: 33. H2N-SDVRD LNALL PAVPS-COOH SEQ ID NO: 34. H2N-DLNAL LPAVP SLGGG-COOH SEQ ID NO: 35. H2N-LLPAV PSLGG GGGCA-COOH SEQ ID NO: 36. H2N-VPSLG GGGGC ALPVS-COOH SEQ ID NO: 37. H2N-GGGGG CALPV SGAAQ-COOH SEQ ID NO: 38. H2N-GCALP VSGAA QWAPV-COOH SEQ ID NO: 39. H2N-PVSGA AQWAP VLDFA-COOH SEQ ID NO: 40. H2N-AAQWA PVLDF APPGA-COOH SEQ ID NO: 41. H2N-APVLD FAPPG ASAYG-COOH SEQ ID NO: 42. H2N-DFAPP GASAY GSLGG-COOH SEQ ID NO: 43. H2N-PGASA YGSLG GPAPP-COOH SEQ ID NO: 44. H2N-AYGSL GGPAP PPAPP-COOH SEQ ID NO: 45. H2N-LGGPA PPPAP PPPPP-COOH SEQ ID NO: 46. H2N-APPPA PPPPP PPPPH-COOH SEQ ID NO: 47. H2N-APPPP PPPPP HSFIK-COOH SEQ ID NO: 48. H2N-PPPPP PHSFI KQEPS-COOH SEQ ID NO: 49. H2N-PPHSF IKQEP SWGGA-COOH SEQ ID NO: 50. H2N-FIKQE PSWGG AEPHE-COOH SEQ ID NO: 51. H2N-EPSWG GAEPH EEQCL-COOH SEQ ID NO: 52. H2N-GGAEP HEEQC LSAFT-COOH SEQ ID NO: 53. H2N-PHEEQ CLSAF TVHFS-COOH SEQ ID NO: 54. H2N-QCLSA FTVHF SGQFT-COOH SEQ ID NO: 55. H2N-AFTVH FSGQF TGTAG-COOH SEQ ID NO: 56. H2N-HFSGQ FTGTA GACRY-COOH SEQ ID NO: 57. H2N-QFTGT AGACR YGPFG-COOH SEQ ID NO: 58. H2N-TAGAC RYGPF GPPPP-COOH SEQ ID NO: 59. H2N-CRYGP FGPPP PSQAS-COOH SEQ ID NO: 60. H2N-PFGPP PPSQA SSGQA-COOH SEQ ID NO: 61. H2N-PPPSQ ASSGQ ARMFP-COOH SEQ ID NO: 62. H2N-QASSG QARMF PNAPY-COOH SEQ ID NO: 63. H2N-GQARM FPNAP YLPSC-COOH SEQ ID NO: 64. H2N-MFPNA PYLPS CLESQ-COOH SEQ ID NO: 65. H2N-APYLP SCLES QPAIR-COOH SEQ ID NO: 66. H2N-PSCLE SQPAI RNQGY-COOH SEQ ID NO: 67. H2N-ESQPA IRNQG YSTVT-COOH SEQ ID NO: 68. H2N-AIRNQ GYSTV TFDGT-COOH SEQ ID NO: 69. H2N-QGYST VTFDG TPSYG-COOH SEQ ID NO: 70. H2N-TVTED GTPSY GHTPS-COOH SEQ ID NO: 71. H2N-DGTPS YGHTP SHHAA-COOH SEQ ID NO: 72. H2N-SYGHT PSHHA AQFPN-COOH SEQ ID NO: 73. H2N-TPSHH AAQFP NHSFK-COOH SEQ ID NO: 74. H2N-HAAQF PNHSF KHEDP-COOH SEQ ID NO: 75. H2N-FPNHS FKHED PMGQQ-COOH SEQ ID NO: 76. H2N-SFKHE DPMGQ QGSLG-COOH SEQ ID NO: 77. H2N-EDPMG QQGSL GEQQY-COOH SEQ ID NO: 78. H2N-GQQGS LGEQQ YSVPP-COOH SEQ ID NO: 79. H2N-SLGEQ QYSVP PPVYG-COOH SEQ ID NO: 80. H2N-QQYSV PPPVY GCHTP-COOH SEQ ID NO: 81. H2N-VPPPV YGCHT PTDSC-COOH SEQ ID NO: 82. H2N-VYGCH TPTDS CTGSQ-COOH SEQ ID NO: 83. H2N-HTPTD SCTGS QALLL-COOH SEQ ID NO: 84. H2N-DSCTG SQALL LRTPY-COOH SEQ ID NO: 85. H2N-GSQAL LLRTP YSSDN-COOH SEQ ID NO: 86. H2N-LLLRT PYSSD NLYQM-COOH SEQ ID NO: 87. H2N-TPYSS DNLYQ MTSQL-COOH SEQ ID NO: 88. H2N-SDNLY QMTSQ LECMT-COOH SEQ ID NO: 89. H2N-YQMTS QLECM TWNQM-COOH SEQ ID NO: 90. H2N-SQLEC MTWNQ MNLGA-COOH SEQ ID NO: 91. H2N-CMTWN QMNLG ATLKG-COOH SEQ ID NO: 92. H2N-NQMNL GATLK GVAAG-COOH SEQ ID NO: 93. H2N-LGATL KGVAA GSSSS-COOH SEQ ID NO: 94. H2N-LKGVA AGSSS SVKWT-COOH SEQ ID NO: 95. H2N-AAGSS SSVKW TEGQS-COOH SEQ ID NO: 96. H2N-SSSVK WTEGQ SNHST-COOH SEQ ID NO: 97. H2N-KWTEG QSNHS TGYES-COOH SEQ ID NO: 98. H2N-GQSNH STGYE SDNHT-COOH SEQ ID NO: 99. H2N-HSTGY ESDNH TTPIL-COOHSEQ ID NO: 100. H2N-YESDN HTTPI LCGAQ-COOHSEQ ID NO: 101. H2N-NHTTP ILCGA QYRIH-COOHSEQ ID NO: 102. H2N-PILCG AQYRI HTHGV-COOHSEQ ID NO: 103. H2N-GAQYR IHTHG VFRGI-COOHSEQ ID NO: 104. H2N-RIHTH GVFRG IQDVR-COOHSEQ ID NO: 105. H2N-HGVFR GIQDV RRVPG-COOHSEQ ID NO: 106. H2N-RGIQD VRRVP GVAPT-COOHSEQ ID NO: 107. H2N-DVRRV PGVAP TLVRS-COOHSEQ ID NO: 108. H2N-VPGVA PTLVR SASET-COOHSEQ ID NO: 109. H2N-APTLV RSASE TSEKR-COOHSEQ ID NO: 110. H2N-VRSAS ETSEK RPFMC-COOHSEQ ID NO: 111. H2N-SETSE KRPFM CAYPG-COOHSEQ ID NO: 112. H2N-EKRPF MCAYP GCNKR-COOHSEQ ID NO: 113. H2N-FMCAY PGCNK RYFKL-COOHSEQ ID NO: 114. H2N-YPGCN KRYFK LSHLQ-COOHSEQ ID NO: 115. H2N-NKRYF KLSHL QMHSR-COOHSEQ ID NO: 116. H2N-FKLSH LQMHS RKHTG-COOHSEQ ID NO: 117. H2N-HLQMH SRKHT GEKPY-COOHSEQ ID NO: 118. H2N-HSRKH TGEKP YQCDF-COOHSEQ ID NO: 119. H2N-HTGEK PYQCD FKDCE-COOHSEQ ID NO: 120. H2N-KPYQC DFKDC ERRFS-COOHSEQ ID NO: 121. H2N-CDFKD CERRF SRSDQ-COOHSEQ ID NO: 122. H2N-DCERR FSRSD QLKRH-COOHSEQ ID NO: 123. H2N-RFSRS DQLKR HQRRH-COOHSEQ ID NO: 124. H2N-SDQLK RHQRR HTGVK-COOHSEQ ID NO: 125. H2N-KRHQR RHTGV KPFQC-COOHSEQ ID NO: 126. H2N-RRHTG VKPFQ CKTCQ-COOHSEQ ID NO: 127. H2N-GVKPF QCKTC QRKFS-COOHSEQ ID NO: 128. H2N-FQCKT CQRKF SRSDH-COOHSEQ ID NO: 129. H2N-TCQRK FSRSD HLKTH-COOHSEQ ID NO: 130. H2N-KFSRS DHLKT HTRTH-COOHSEQ ID NO: 131. H2N-SDHLK THTRT HTGKT-COOHSEQ ID NO: 132. H2N-KTHTR THTGK TSEKP-COOHSEQ ID NO: 133. H2N-RTHTG KTSEK PFSCR-COOHSEQ ID NO: 134. H2N-GKTSE KPFSC RWPSC-COOHSEQ ID NO: 135. H2N-EKPFS CRWPS CQKKF-COOHSEQ ID NO: 136. H2N-SCRWP SCQKK FARSD-COOHSEQ ID NO: 137. H2N-PSCQK KFARS DELVR-COOHSEQ ID NO: 138. H2N-KKFAR SDELV RHHNM-COOHSEQ ID NO: 139. H2N-RSDEL VRHHN MHQRN-COOHSEQ ID NO: 140. H2N-LVRHH NMHQR NMTKL-COOHSEQ ID NO: 141. H2N-HNMHQ RNMTK LQLAL-COOH

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What is claimed is:
 1. An isolated WT-1 peptide consisting ofan aminoacid sequence selected from among AILDFLLLQ (SEQ ID NO:147), RQRPHPGAL(SEQ ID NO:142), GALRNPTAC (SEQ ID NO:143), THSPTHPPR (SEQ ID NO:146),ASGSEPQQM (SEQ ID NO:151), WNQMNLGATLK (SEQ ID NO:173), PGCLQQPEQQG (SEQID NO:149), and LDFAPPGASAY (SEQ ID NO:156).
 2. An isolated WT-1 peptideconsisting ofan amino acid sequence selected from among PLPHFPPSL (SEQID NO:144), HFPPSLPPT (SEQ ID NO:145), PGCLQQPEQ (SEQ ID NO:148),KLGAAEASA (SEQ ID NO:150), LLAAILDFL (SEQ ID NO:184), CLQQPEQQGV (SEQ IDNO:185) and ALRNPTACPL (SEQ ID NO:191).
 3. An isolated WT-1 peptideconsisting of an amino acid sequence selected from amongRDLNALLPAV (SEQID NO:152), GGCALPVSGA (SEQ ID NO:153), GAAQWAPVL (SEQ ID NO:154),LDFAPPGAS (SEQ ID NO:155), SAYGSLGGP (SEQ ID NO:157), PAPPPPPPP (SEQ IDNO:158), ACRYGPFGP (SEQ ID NO:159), SGQARMFPN (SEQ ID NO:160), PSCLESQPA(SEQ ID NO:162), NQGYSTVTF (SEQ ID NO:163), HHAAQFPNH (SEQ ID NO:164),HSFKHEDPM (SEQ ID NO:165), CHTPTDSCT (SEQ ID NO:166), CTGSQALLL (SEQ IDNO:167), TDSCTGSQA (SEQ ID NO:168), RTPYSSDNL (SEQ ID NO:169),NLYQMTSQLE (SEQ ID NO:170), WNQMNLGAT (SEQ ID NO:171), WNQMNLGATLK (SEQID NO:173), CMTWNQMNLGATLKG (SEQ ID NO:174), NLGATLKGV (SEQ ID NO:175),LGATLKGVAA (SEQ ID NO:176), TLGVAAGS (SEQ ID NO:177), GYESDNHTT (SEQ IDNO:178), FMCAYPGCNK (SEQ ID NO:179), KRPFMCAYPGC (SEQ ID NO:180),RKFSRSDHL (SEQ ID NO:181), LKTHTTRTHT (SEQ ID NO:182), NMHQRNHTKL (SEQID NO:183), QARMFPNAPY (SEQ ID NO:190), ALRNPTACPL (SEQ ID NO:191) andAPVLDFAPPGASAYG (SEQ ID NO:193).
 4. An isolated WT-1 peptide consistingofan amino acid sequence selected from among SEQ ID NO:1-141.
 5. Anisolated WT-1 peptide consisting of 8-30 amino acids comprising an aminoacid sequence selected from SEQ ID NO: 142, 143, 144, 145, 146, 147,148, 149, 150, 151, 184 and
 185. 6. An isolated WT-1 peptide consistingof 16-30 amino acids comprising an amino acid sequence selected from SEQID NO:1-141.
 7. The isolated WT-1 peptide of any one of claims 1-6,wherein said isolated WT-1 peptide binds to an HLA class I molecule, anHLA class II molecule, or the combination thereof.
 8. A pharmaceuticalcomposition comprising a peptide of any one of claims 1-6 and apharmaceutically acceptable carrier, vehicle or excipient.
 9. A vaccinecomprising (a) one or more isolated WT-1 peptides of claims 1-6 and (b)an adjuvant or a carrier.
 10. The vaccine of claim 9, wherein saidadjuvant is QS21, Freund's incomplete adjuvant, aluminum phosphate,aluminum hydroxide, BCG, alum, a growth factor, a cytokine, a chemokine,an interleukin, Montanideor GM-CSF.
 11. A method of treating a subjectwith a WT-1-expressing cancer or reducing an incidence of aWT-1-expressing cancer, or its relapse, the method comprisingadministering to said subject the vaccine of claim 9, thereby treating asubject with a WT-1-expressing cancer, reducing an incidence of aWT-1-expressing cancer or its relapse therein.
 12. The method of claim11, wherein said WT-1-expressing cancer is a leukemia, a desmoplasticsmall round cell tumor, a gastric cancer, a colon cancer, a lung cancer,a breast cancer, a germ cell tumor, an ovarian cancer, a uterine cancer,a thyroid cancer, a liver cancer, a renal cancer, a Kaposi's sarcoma, asarcoma, a hepatocellular carcinoma, a Wilms' tumor, an acutemyelogenous leukemia (AML), a myelodysplastic syndrome (MDS), or anon-small cell lung cancer (NSCLC).
 13. A method of inducing theformation and proliferation of CTL specific for cells of aWT-1-expressing cancer, the method comprising administering to saidsubject the vaccine of claim 9, thereby inducing the formation andproliferation of CTL specific for cells of a WT-1-expressing cancer. 14.The method of claim 13, wherein said WT-1-expressing cancer is aleukemia, a desmoplastic small round cell tumor, a gastric cancer, acolon cancer, a lung cancer, a breast cancer, a germ cell tumor, anovarian cancer, a uterine cancer, a thyroid cancer, a liver cancer, arenal cancer, a kaposi's sarcoma, a sarcoma, a hepatocellular carcinoma,a Wilms' tumor, an acute myelogenous leukemia (AML), a myelodysplasticsyndrome (MDS), or a non-small cell lung cancer (NSCLC).
 15. Acomposition comprising (a) an antigen-presenting cell and (b) a peptideof any one of claims 1-6.
 16. The method of any one of claims 11-14wherein the cancer is mesothelioma.